TWI711633B - Tumor antigen peptide - Google Patents

Abstract

The invention is directed to providing a tumor antigen peptide which is specifically expressed in a cancer cell and a cancer stem cell, and to providing a pharmaceutical composition containing such a tumor antigen peptide as an effective ingredient, wherein the tumor antigen peptide is beneficial for preventing and/or treating a cancer.

For the aforementioned purpose, the problems are addressed in the invention by providing the follows: a partial peptide derived from BORIS (Brother of Regulator of Imprinted Sites) belonging to isoform A or C or belonging to isoform subgroup 5 or 6; a polynucleotide encoding the aforementioned peptide; a pharmaceutical composition containing said peptide and/or polynucleotide as the effective ingredient; and a cancer prophylactic and/or therapeutic agent which is characterized in that the agent can induce cytotoxic T cells.

Description

Tumor antigen peptide

The present invention relates to a tumor antigen peptide derived from BORIS, etc., which is useful as a preventive and/or therapeutic agent for cancer.

The therapeutic effects of anticancer agents developed so far are not sufficient, and the probability that cancer can be cured by treatment with only anticancer agents is extremely low. The reason for this may be that the conventional therapeutic agents failed to selectively target cells that form the root of cancer tissues. In recent years, the existence of cancer stem cells has been reported as the "cells that form the root of cancer tissue." Cancer stem cells are cells that exist in a very small proportion of cancer cells and have high tumor-forming ability, self-replication ability and differentiation ability. They are considered to be the causal cells involved in the occurrence, recurrence and metastasis of cancer. Therefore, if cancer stem cells can be targeted, it is expected that there is a high possibility that cancer proliferation, recurrence, and metastasis can be effectively suppressed. That is, the detection technology of cancer stem cells and the development of novel therapeutic agents that target cancer stem cells have become important issues for cancer medicine.

On the other hand, in the elimination of tumor cells or virus-infected cells derived from organisms, cellular immunity, especially cytotoxic T lymphocyte (CTL) is responsible for the Important job. In the elimination of tumor cells, CTL recognizes the antigen peptides (tumor antigen peptides) and MHC (Major Histocompatibility Complex) type I antigens on tumor cells (called in humans) HLA (human leukocyte antigen, type I antigen) complex, which attacks and destroys tumor cells. Tumor antigen peptides are proteins (tumor antigen proteins) that are specifically expressed in tumor cells. After being synthesized in the cells, they are produced by being broken down in the cells by proteolytic enzymes. The generated tumor antigen peptide binds to MHC type I antigen (HLA type I antigen) in the endoplasmic reticulum to form a complex, and is transported to the cell surface for antigen presentation. In this way, tumor-specific CTLs recognize complexes containing antigen peptides presented by antigens, and attack tumor cells by cytotoxicity or lymphoid production, etc., and exhibit anti-tumor effects. With the elucidation of such a series of effects, by using tumor antigen proteins or tumor antigen peptides as so-called cancer immunotherapy agents (cancer vaccines), treatments for enhancing cancer-specific CTLs in cancer patients are continuously being developed.

The BORIS (Brother of the Regulator of Imprinted Sites) gene is a homologue of the CTCF gene. It is one of the two peptide regions encoding the N-terminal peptide region and the C-terminal peptide region. There are 11 zinc finger areas. BORIS is known to function as a general transcription factor like repressors and activators of various gene expressions. It is also known to be expressed in various tumor cells, especially cancer stem cells. Also, there are reports It is pointed out that the zinc finger region of BORIS has high identity with the CTCF gene. In BORIS, there are 23 isoforms classified into 6 subtypes, all of which are splice variants (non- Patent Document 1). In addition, BORIS has attracted attention as a candidate target for cancer diagnosis and treatment because it does not express in normal tissues other than testicles. There are reports that specific antibodies to each subtype of BORIS or siRNA used to hinder the expression of BORIS genes, etc. ( Patent documents 1, 2, etc.). There are also reports that analyze the sequence of the BORIS protein and predict the sequence restricted to HLA-A0201 from the sequence. One of the sequences can induce CTL, which will actually specifically identify the antigen presented Cells (Non-Patent Document 2).

[Prior Technical Literature] (Patent Document)

Patent Document 1: International Publication No. 2008/028066 Booklet

Patent Document 2: US Patent Application Publication No. 2009/0169613

(Non-patent literature)

Non-Patent Document 1: Pugacheva et al., PLoS ONE 5(11): e13872

Non-Patent Document 2: Romagnoli et al., Rapporti ISTISAN. 2006;06(50), 36-40

The purpose of the present invention is to provide a tumor antigen peptide derived from BORIS, particularly a specific tumor antigen peptide of BORIS isoforms or subtypes; and, to provide a tumor antigen peptide containing this tumor antigen peptide as an effective ingredient Medical compositions, etc., which are beneficial to the prevention and/or treatment of cancer, especially medical compositions that specifically treat cancer stem cells.

The inventors of the present case studied BORIS as a tumor antigen for cancer vaccine therapy and found that BORIS specifically exhibits specific subtypes even among cells that exhibit stem cell properties in cervical cancer or ovarian cancer. BORIS. After further research based on this insight, it was found that by inducing cytotoxic T cells (CTL), it can identify BORIS cells that exhibit specific isoforms or subtypes, and can treat many cancer cells and/or Cancer stem cells are the result of further research and the present invention has been completed.

That is, the present invention relates to the following disclosures:

[1] A method for inducing cytotoxic T cells. The cytotoxic T cells specifically identify cells expressing the BORIS gene. The BORIS gene belongs to isoforms A or C, or subtype 5 or 6, the method It includes contacting any of the following (a) or (b) with peripheral blood lymphocytes: (a) Polypeptide, which is a partial peptide of the aforementioned BORIS protein, with 8-20 amine groups Long acidity and ability to bind human leukocyte antigen (HLA); (b) Polynucleotide which encodes at least one of the polypeptides described in (a) above.

[2] The method for inducing cytotoxic T cells as described in [1], wherein the method is performed in vitro.

[3] A polypeptide used in the method described in [1] or [2] is a partial peptide of the polypeptide represented by sequence number 1 or sequence number 2, which is 8-20 amines The base acid is long and has HLA binding ability.

[4] The polypeptide as described in [3], which has an amino acid length of 8-11.

[5] The polypeptide according to [3] or [4], which is a partial peptide of the polypeptide represented by SEQ ID NO:1.

[6] The polypeptide according to [5], which is represented by SEQ ID NO: 3, SEQ ID No. 4, SEQ ID No. 5, or SEQ ID No. 72.

[7] The polypeptide according to [3] or [4], which is a partial peptide of the polypeptide represented by SEQ ID NO: 2.

[8] The polypeptide described in [7], which is represented by SEQ ID NO: 10.

[9] A polypeptide, which is used in a method such as [1] or [2], and has HLA-A11 antigen binding ability.

[10] The polypeptide of [9] is represented by SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, or SEQ ID No. 72.

[11] A polypeptide which is used in the method described in [1] or [2] and has HLA-A2 antigen binding ability, which is based on sequence number 4, sequence number 5, sequence number 10, and sequence number 47 or serial number 57 to indicate.

[12] A polypeptide which is used in the method described in [1] or [2] and has HLA-A24 antigen binding ability.

[13] Polypeptides such as [12] are represented by SEQ ID NO: 3 or 10.

[14] A polypeptide in which one or more amino acids are added, deleted, or substituted in the polypeptide described in any one of [3] to [13].

[15] A polynucleotide used in the method described in [1] or [2] to encode the polypeptide described in any one of [3] to [14].

[16] A cytotoxic T cell inducer comprising at least one of the polypeptides described in any one of [3] to [14] as an active ingredient.

[17] A pharmaceutical composition comprising the cytotoxic T cell inducer described in [16] as an effective ingredient.

[18] A composition for treating cancer stem cells, which contains the cytotoxic T cell inducer described in [16] as an active ingredient.

[19] A composition for the prevention and/or treatment of cancer, which comprises the cytotoxic T cell inducer described in [16] as an active ingredient.

[20] A composition for the prevention and/or treatment of cancer, comprising as an active ingredient cytotoxic T cells induced by the method described in [1] or [2].

[21] The composition for the prevention and/or treatment of cancer as described in [19] or [20], wherein the cancer is cancer or lung cancer in female organs.

[22] A performance vector containing the polynucleotide as described in [15].

[23] A pharmaceutical composition for the treatment or prevention of cancer, which contains either the polynucleotide described in [15] or the expression vector described in [22] as an active ingredient.

[24] An HLA-tetramer comprising the polypeptide described in any one of [3] to [14] and HLA.

[25] A method for producing antigen-presenting cells, comprising the steps of contacting the following (a) or (b) with cells having antigen-presenting ability in a test tube: (a) any of [3] to [14] One of the polypeptide (b) polynucleotides, which encodes at least one of the polypeptides described in (a) above.

[26] An antibody that specifically binds to at least a part of the polypeptide represented by SEQ ID No. 1 or SEQ ID No. 2.

[27] A kit for detecting BORIS protein, comprising the antibody described in [26].

According to the present invention, it is possible to provide: a method for inducing cytotoxic T cells (CTL), the CTL will recognize a specific isoform or subtype of BORIS; a tumor antigen peptide, which is used as a CTL for the method The inducer is beneficial; and, a pharmaceutical composition containing the peptide as an effective ingredient, etc., which is beneficial to the prevention and/or treatment of cancer. Particularly among cancers, there is BORIS that exhibits specific subtypes among cells with stem cell-like properties (that is, cancer stem cells). Therefore, the therapeutic agent of the present invention can selectively treat cancer stem cells. Becomes possible.

Figure 1 is a photograph of a spheroid formed by the spheroid formation analysis method.

Figure 2 is a graph showing the relative expression levels of stem cell genes SOX2, NANOG, and Oct3/4 in cervical cancer cell lines CaSki and TCS cell lines.

Figure 3 is a graph showing the performance of BORIS in normal tissues; (a) is a graph showing the results of RT-PCR, and (b) is a graph showing the relative expression level.

Figure 4 is a graph showing the relative expression levels of BORIS in the clumped cells and spheroid cells of various cervical cancer cell lines.

Figure 5 is a graph showing the relative expression levels of BORIS in various cancer cells.

Figure 6 is a graph showing the comparison of the expression levels of each BORIS subtype of the mass cell and the spheroid cell among the cervical cancer cell lines CaSki and MS751 cell lines.

Figure 7 is a graph showing a comparison of the expression levels of each BORIS subtype of the ovarian cancer cell lines TOV21G and smov2 cell lines, the mass cell and the spheroid cell.

Figure 8-1 is a photograph showing the results of spheroid formation analysis when a large number of BORIS subtypes were expressed in the cervical cancer cell line, that is, the TCS cell line.

Figure 8-2 is a graph showing the results of spheroid formation analysis when a large number of BORIS subtypes were expressed in the cervical cancer cell line, that is, the TCS cell line.

Figure 9-1 is a photograph showing the results of analysis of spheroid formation when a large number of BORIS subtypes were expressed in the cervical cancer cell line, namely the SKG-IIIb cell line.

Figure 9-2 is a graph showing the results of spheroid formation analysis when a large number of BORIS subtypes were expressed in the cervical cancer cell line, that is, the SKG-IIIb cell line.

Figure 10 is a graph showing the results of HLA-A02 binding analysis of candidate HLA-binding peptides extracted from BORIS sf6 specific sequences.

Figure 11 is a graph showing the results of the folding test of the HLA-A*24:02-binding BORIS-specific CTL epitope candidate peptide; this graph shows the HLA deduced from the peak area of the HLA-monomer -Amount of monomer formation.

Figure 12-1 shows the first stage of analysis in which the sample collected from the sample number A24-38 is co-cultured with the RMM peptide, and then analyzed by a flow cytometer to react with the HLA-tetramer reagent. The result graph.

Figure 12-2 shows the second stage of analysis in which the samples collected from the sample number A24-38 are co-cultured with RMM peptides and then analyzed by flow cytometry and reacted with HLA-tetramer reagents. Result graph: In the first stage of analysis, because CTLs were detected in the second, fourth, eighth, and 9th lines, in the second stage of analysis, these lines were analyzed.

Figure 13 is a diagram showing the results of functional analysis of the function of RMM peptide-specific CTL using RMM-Tet; when stimulated with RMM peptide, it was found that IFNγ was specifically produced by RMM peptide.

Figure 14-1 is a graph showing the comparison of mRNA expression (a) and cell proliferation (b) in CaSki cell lines transfected with siRNA.

Figure 14-2 is a microscope image of CaSki cells transfected with siRNA.

Figure 15 is a chart comparing the gene expression of stem cells in CaSki cell lines transfected with siRNA.

Figure 16-1 is a graph comparing the sphere-forming ability of CaSki cell line (a) and MS751 cell line (b) transfected with siRNA.

Figure 16-2 is a microscope image of each cell line transfected with siRNA.

Figure 17 is a graph showing the comparison of the radiation tolerance of cell lines transfected with siRNA.

Figure 18 shows the Kaplan-Meier survival curve (Kaplan-Meier survival curve) showing that if BORIS expression is high, the survival rate will be significantly reduced.

Figure 19-1 is a graph showing the comparison of the expression levels of each BORIS subtype of the mass cells and the spheroid cells among the small cell lung cancer cell lines SBC1, SBC3, SBC5, and Lc817 cell lines.

Figure 19-2 is a graph showing the comparison of the expression levels of each BORIS subtype of the mass cell and the spheroid cell among the non-small cell lung cancer cell lines Lu99A and 86-2 cell lines.

Figure 19-3 is a graph showing a comparison of the expression levels of each BORIS subtype of the mass cell and the spheroid cell among the lung squamous cell lines LK2, EBC1, and Sq1 cell lines.

Figure 19-4 is a graph showing the comparison of the expression levels of each BORIS subtype of the mass cells and the spheroid cells among the lung adenocarcinoma cell lines A549, LHK2, LHK2-SOX2, and PC3 cell lines.

Figure 19-5 is a graph showing the comparison of the expression levels of each BORIS subtype of the mass cell and the spheroid cell among the primary cultured lung adenocarcinoma cells Primary3, Primary4, Primary5, and Primary7 cells.

Figure 20 is a diagram showing the results of the first stage analysis of the sample collected from the sample number A2-34, co-cultured with KLL peptide, and analyzed with a flow cytometer to analyze the reaction with the HLA-tetramer reagent .

Figure 21 is a diagram showing the results of the second stage analysis of the sample collected from the sample number A2-34, co-cultured with KLL peptide, and analyzed by a flow cytometer for the reaction with the HLA-tetramer reagent ; Since CTL was detected in lines 5 and 11 in the first stage of analysis, these lines were analyzed in the second stage of analysis.

Figure 22 shows the results of the first stage of analysis in which the sample collected from the sample number A2-29 was co-cultured with the LLF peptide, and then analyzed with a flow cytometer to react with the HLA-tetramer reagent. .

Figures 23-1 and 23-2 show the samples collected from the sample number A2-29, co-cultured with the LLF peptide, and analyzed by a flow cytometer to react with the HLA-tetramer reagent The result graph of the second stage of analysis; because in the first stage of analysis, in the first row, 2, 4, 5, 6, CTL was detected in 7, 8, 9, 10, and 11, so these lines are analyzed in the second stage of analysis.

Figure 24 is a graph showing the results of analyzing the reaction with the HLA-tetramer reagent using a flow cytometer after co-culturing the samples collected from the specimen number A2-S1 with LLF peptide-presenting cells.

Figure 25 is a graph showing the results of analyzing the IFNγ production capacity of the induced CTL by using ELISPOT to analyze the IFNγ production capacity of the induced CTL by co-cultivating the samples with the sample numbers A2-S1, A2-S2, A2-S3 and LLF peptide presenting cells .

Figure 26 shows the samples collected from the sample number A2-S1, co-cultured with LLF peptide-presenting cells, and then the induced CTLs are monocultured and amplified, and then analyzed and analyzed by a flow cytometer. HLA-tetramer reagent reaction result graph.

Figure 27 shows the analysis of the production capacity of IFNγ by ELISPOT after co-cultivating the samples with LLF peptide presenting cells collected from samples No. A2-S1, after the induced CTLs were monocultured and amplified The result graph.

Figure 28 shows the samples collected from the specimens numbered A2-S1, after co-cultivation with LLF peptide presenting cells, the induced CTLs were cloned and amplified, and then the cytotoxicity was analyzed by the LDH killing assay. Activity result graph.

Figure 29 shows that the samples collected from the specimen number A24-S4 or specimen number A2-S5 are co-cultured with RMM peptide presenting cells and then used The flow cytometer analyzes the result graph of the reaction with the HLA-tetramer reagent.

Figure 30 is a graph showing the results of analyzing the IFNγ production capacity of CTLs induced by ELISPOT after co-cultivation with RMM peptide presenting cells collected from samples with sample numbers A24-S4 or A2-S5 .

Figure 31 shows the samples collected from the sample number A24-S4 and co-cultured with RMM peptide-presenting cells. After the induced CTLs were monocultured and amplified, they were analyzed and analyzed by a flow cytometer. HLA-tetramer reagent reaction result graph.

Figure 32 shows the samples collected from the samples Nos. A2-S5, after co-cultivation with RMM peptide presenting cells, after the induced CTL was monocultured and amplified, analyzed and analyzed by flow cytometry HLA-tetramer reagent reaction result graph.

Figure 33 is a graph showing the results of the folding test of the HLA-A*02:01 restricted, BORIS-specific CTL epitope candidate peptide; this graph is deduced from the peak area of the HLA-monomer HLA-monomer formation amount.

Figure 34 is a diagram showing the results of the first stage of analysis in which the sample collected from the sample number A2-29 was co-cultured with the VLE peptide, and then analyzed with a flow cytometer to react with the HLA-tetramer reagent. .

Figure 35 is a diagram showing the results of the second stage analysis of the sample collected from the sample number A2-29, co-cultured with the VLE peptide, and analyzed by a flow cytometer to react with the HLA-tetramer reagent ; At the first order In the segment analysis, because the CTL was detected in the 10th line, in the second stage of the analysis, the analysis was performed on the 10th line.

Figure 36 is a diagram showing the results of the first stage analysis of the sample collected from the sample number A2-27, co-cultured with the VLE peptide, and analyzed by a flow cytometer to react with the HLA-tetramer reagent .

Figure 37 is a diagram showing the results of the second stage analysis of the sample collected from the sample number A2-27, co-cultured with the VLE peptide, and analyzed by the flow cytometer to react with the HLA-tetramer reagent ; In the first stage of analysis, because the CTL was detected in the third row, in the second stage of analysis, the third row was analyzed.

Figure 38 is a diagram showing the results of the first stage analysis of the sample collected from the sample number A2-34, co-cultured with the VLE peptide, and analyzed by the flow cytometer to react with the HLA-tetramer reagent .

Figure 39 is a diagram showing the results of the second stage analysis of the sample collected from the sample number A2-34, co-cultured with the VLE peptide, and analyzed by the flow cytometer to react with the HLA-tetramer reagent ; In the first stage of analysis, because the CTL was detected in the 4th line, in the second stage of the analysis, the 4th line was analyzed.

Figure 40 is a diagram showing the results of the first stage analysis of the sample collected from the sample number A2-29, co-cultured with KLA peptide, and analyzed by a flow cytometer with the HLA-tetramer reagent. .

Figure 41 is a diagram showing the results of the second stage analysis of the sample collected from the sample number A2-29, co-cultured with KLA peptides, and analyzed by flow cytometry with the HLA-tetramer reagent. ;in In the first stage of analysis, because the CTL was detected in the second, fifth, and 11th lines, in the second stage of the analysis, the second, fifth, and 11th lines were analyzed.

Figure 42 is a diagram showing the results of the first stage analysis of the sample collected from the sample number A2-29, co-cultured with the VLT peptide, and analyzed by a flow cytometer with the HLA-tetramer reagent. .

Figure 43 is a diagram showing the results of the second stage analysis in which the sample collected from the sample number A2-29 was co-cultured with the VLT peptide, and then analyzed with a flow cytometer to react with the HLA-tetramer reagent. ; In the first stage of analysis, because the CTL was detected in the 7th and 9th lines, in the second stage of the analysis, the 7th and 9th lines were analyzed.

Figure 44 is a graph showing the results of functional analysis of the function of KLA peptide-specific CTL using KLA-Tet; when stimulated with KLA peptide, it can be seen that the KLA peptide specifically detects CD107a.

Figure 45 is a diagram showing the results of functional analysis of the function of VLT peptide-specific CTL using VLT-Tet; when stimulated with VLT peptide, it can be seen that the VLT peptide specifically induces CD107a.

Figure 46 is a graph showing the results of the folding test of the HLA-A*11:01 restricted, BORIS-specific CTL epitope candidate peptide; this graph shows the peak area of the HLA monomer.

Figure 47 shows that the samples collected from the sample number *11-13 are co-cultured with SVL peptides, NTH peptides, KQL peptides, and GLI peptides, and then analyzed by flow cytometry with HLA-4 The result of the first stage analysis of the reaction of the polymer reagent.

Figure 48 shows that the samples collected from the sample number *11-13 were co-cultured with SVL peptides, NTH peptides, KQL peptides, and GLI peptides, and then analyzed by flow cytometry with HLA-4 Figure of the results of the second stage analysis of the reaction of the polymer reagent; in the first stage analysis, since the CTL was detected in the first line, the second stage analysis was performed on the first line.

Figure 49 shows the samples collected from the sample number *11-13, co-cultured with SVL peptides, NTH peptides, KQL peptides, and GLI peptides, and then analyzed by flow cytometry and HLA-tetramerization Figure of the results of the third stage analysis of the reaction of the body reagent; in the second stage of analysis, since CTL was detected in the first row of well B, in the third stage of analysis, the first row of well B was analyzed .

Figure 50 shows that the samples collected from the sample number *11-13 are co-cultured with SLA peptides, CSY peptides, TVY peptides, and TVL peptides, and then analyzed by flow cytometry with HLA-4 The result of the first stage analysis of the reaction of the polymer reagent.

Figure 51 shows that the samples collected from the sample number *11-13 are co-cultured with SLA peptides, CSY peptides, TVY peptides, and TVL peptides, and then analyzed by flow cytometry with HLA-4 Figure of the results of the second stage analysis of the reaction of the polymer reagent; in the first stage analysis, since the CTL was detected in the 12th line, the second stage analysis was performed on the 12th line.

Figure 52 shows the samples collected from the sample number *11-13 and co-cultured with SLA peptides, CSY peptides, TVY peptides, and TVL peptides. Then, use a flow cytometer to analyze the results of the third-stage analysis of the reaction with the HLA-tetramer reagent; in the second-stage analysis, since CTL was detected in well E in row 12, In the third stage of analysis, the hole E in row 12 is analyzed.

Figure 53 shows that the samples collected from the sample number *11-16 are co-cultured with RMS peptides, GTM peptides, AAA peptides, and KLLF peptides, and then analyzed by flow cytometry with HLA-4 The result of the first stage analysis of the reaction of the polymer reagent.

Figure 54 shows that the samples collected from the sample number *11-16 were co-cultured with RMS peptides, GTM peptides, AAA peptides, and KLLF peptides, and then analyzed by flow cytometry with HLA-4 Figure of the results of the second stage analysis of the reaction of the polymer reagent; in the first stage analysis, since CTL was detected in the 7th and 11th rows, the second stage of the analysis is for the 7th and 11th rows Analyze.

Figure 55 shows that the samples collected from the sample number *11-16 are co-cultured with the RMS peptide, GTM peptide, AAA peptide, and KLLF peptide, and then analyzed by a flow cytometer with HLA-4 Figure of the results of the third-stage analysis of the polymer reagent reaction; in the second-stage analysis, since CTL was detected in the hole E in the 7th row and the hole H in the 11th row, the third-stage analysis was The hole E in the 7th row and the hole H in the 11th row are analyzed.

Figure 56 shows the results of Western blotting using 293T cell extract. This cell extract is a 293T cell extract after briefly forced expression of BORIS sf5 and BORIS sf6 with Myc Tag attached. Take the liquid; no matter which specific antibody is used, the band can be confirmed at the same position as when using Myc Tag antibody, so it shows the specificity of these antibodies.

Figure 57 shows the results of immunostaining of lung cancer tissue slices using BORIS sf5 specific antibody; even the same lung cancer tissue, BORIS sf5 is confirmed to be positive and negative; where (a) means Negative, (b) is a positive staining image, and compared to (b), the cells stained in brown are almost invisible in (a).

Hereinafter, the present invention will be described in detail.

(1) Polypeptide of the present invention

The "antigenic determinant peptide" in the present invention means a polypeptide that binds to MHC (HLA in humans) and presents an antigen on the cell surface. The epitope peptides include CTL epitope peptides, which bind to MHC type I and are presented by antigens, and are recognized by CD8-positive T cells; and helper cell epitope peptides, which interact with MHC Type II binds and is presented by antigen, and is recognized by CD4-positive T cells.

Among the epitope peptides, polypeptides derived from proteins expressed specifically or excessively in tumor cells are particularly called tumor antigen peptides. The so-called antigen presentation is the combination of polypeptide present in the cell with MHC, and this MHC/antigen peptide complex is localized on the cell surface The phenomenon is called. As mentioned above, it is known that after the antigens presented on the cell surface are recognized by T cells etc., cellular immunity or humoral immunity will be activated. Due to the antigens presented by MHC type I, along with the activation of cellular immunity, It will be recognized by the T cell receptors of naive T cells and induce naive T cells to CTLs with cytotoxic activity. Therefore, as a tumor antigen peptide for immunotherapy, it is better to bind to MHC type I, and A polypeptide presented by an antigen.

On the other hand, antigen proteins taken up by antigen-presenting cells such as dendritic cells are displayed on the surface of antigen-presenting cells by binding to MHC type II, and are recognized by CD4-positive T cells, and finally can induce Helper T cells that activate cellular immunity or humoral immunity. Since helper T cells not only have cytotoxic activity like CTL, they also play an important role in the maintenance of CTL activity and survival. Therefore, as tumor antigen peptides for immunotherapy, they are also preferably combined with MHC type II. And the polypeptide presented by the antigen. It is known that the polypeptide bound to MHC type I is about 8-11 amino acids long, and the polypeptide bound to MHC type II is about 12-20 amino acids long.

In the present invention, "tumor" includes benign tumors and malignant tumors (carcinoma, malignant neoplasm). Cancer includes tumors of hematopoietic organs, epithelial malignancies (carcinoma) and non-epithelial malignancies (sarcoma).

In the present invention, the term "BORIS" alone means that the brother of the imprint site regulator (Brother of the Regulator of Imprinted Sites) genes or their expression products are either mRNA or protein. In the expression control of the BORIS gene, three kinds of promoters (called promoter A, promoter B, and promoter C in sequence from the upstream promoter) are known to participate in it. It is controlled to be roughly divided into 3 isoforms (corresponding to each promoter, respectively called isoform A, isoform B, and isoform C). And each isoform is further distinguished into plural splicing variants due to the splicing mode received during transcription. From this, BORIS is known to have a total of 23 isoforms of 6 isoforms A (A1~A6), 8 isoforms B (B0~B7), and 9 isoforms C (C1~C9). Structure. For example, the BORIS C1 isoform has a sequence represented by sequence number 76.

As mentioned above, BORIS is a homolog of CTCF also known as 11-zinc finger protein. BORIS protein has an N-terminal peptide region and a C-terminal region on the N-terminal side and C-terminal side of the 11 zinc finger regions. The structure of the peptide region. In the N-terminal peptide region, there are products with 24 amino acid lengths, 53 amino acid lengths and 258 amino acid lengths according to the isoforms, but those with the same length have a high degree of sequence retention. There are products of various lengths in the C-terminal peptide region, and their sequences are also different. BORIS is classified into 6 subtypes based on the sequence of the C-terminal peptide region (subtypes 1 to 6 are abbreviated as sf1 to 6 in this specification). Therefore, the C-terminal sequence of each subtype has a unique sequence in each subtype, and is highly retained among isoforms belonging to the same subtype.

The polypeptide of the present invention is a BORIS expressing a specific isoform or subtype, specifically, a cell expressing a BORIS cell belonging to isoform A or C, or subtype 5 or 6. On the surface, by the antigen presenter. Therefore, the polypeptide of the present invention is a partial peptide of BORIS belonging to isoforms A or C, or subtype 5 or 6. It is a polypeptide with 8-20 amino acids in length and has HLA binding ability. . Among cancer cells containing cancer stem cells, most cancer cells expressing these BORIS are present. Therefore, the polypeptide of the present invention is useful in cancer immunotherapy.

The polypeptide of the present invention, in one aspect, the sequence unique to BORIS sf6 is the sequence number 1 or the sequence unique to BORIS sf5 is the partial peptide of the polypeptide represented by the sequence number 2. It contains MHC, especially peptides that bind to HLA, preferably contains MHC, especially peptides that are presented by antigens by HLA, and more preferably contains MHC, especially peptides that are presented by HLA by antigens and can induce CTL Polypeptides of peptides. Although there are several types of HLA, the polypeptide of the present invention is preferably capable of binding to HLA type I, more preferably capable of binding to HLA-A24, HLA-A11 or HLA-A02, and even more preferably capable of binding It binds to more than two HLAs among HLA-A02, HLA-A11 and HLA-A24. In another aspect, a polypeptide capable of binding to HLA type II is also preferred.

For example, when MHC is HLA type I, it is known that most of the antigens presented by HLA type I molecules are broken down by the proteasome in the cytoplasm and then transferred to TAP (transporter in antigen processing). ) Transfer, combine with the complex of HLA type I molecules and β 2-microglobulin in the rough endoplasmic reticulum and collected in TAP, and transport them to the cell surface through exocytosis through high-gei bodies. Therefore, the polypeptide of the present invention can also be processed before binding to MHC to produce peptides such as epitope peptides as a result of these treatments, which are also included in the polypeptide of the present invention. For example, by fusing HSP70 and HSP90, or gp96, which act on the aforementioned sequence of antigen presentation pathways, with the target peptide or protein, the antigen presentation can be performed efficiently. In one aspect, the polypeptide of the invention is fused with a companion protein that acts on the antigen presentation pathway.

In addition, the epitope peptide of the present invention can also be modified by various modifications that can be easily introduced into the body. As an example of various modifications that can be easily introduced into a living body, the PT (Protein Transduction) domain of HIV can be cited. The PT domain of HIV is a peptide composed of amino acids 49 to 57 of the Tat protein. By adding such modifications to the N-terminus and/or C-terminus of the target protein or peptide, the target protein or peptide can be easily introduced into the cell.

As mentioned above, since the polypeptide of the present invention can be processed before binding to MHC, the length of the amino acid is not particularly limited as long as it contains the amino acid sequence of the epitope peptide. However, the polypeptide of the present invention is preferably an epitope peptide, so the amino acid length is preferably about 8-20 amino acids, and more preferably about 8-11 amino acids. , More preferably about 8-10 amino acids.

In a preferred aspect, the polypeptide of the present invention is a partial peptide of the polypeptide represented by SEQ ID NO:1, is a polypeptide of 8-11 amino acids long, and has HLA binding ability.

In another preferred aspect, the polypeptide of the present invention is a partial peptide of the polypeptide represented by SEQ ID NO: 2 and is a polypeptide with 8-11 amino acids long and having HLA binding ability.

Whether the polypeptide has "HLA binding ability" can be easily investigated using methods known in this technical field. The method is not limited to this. For example, as described in WO2010/50190, a monoclonal antibody against HLA is used to reduce the amount of HLA expressed on the cell surface (that is, binding to polypeptide). The amount of HLA) used as fluorescence intensity to observe HLA binding analysis; in Kim et al., Methods Mol Biol. 2013; 960: 447-59, etc., the binding analysis using BIAcore surface plasmon resonance (SPR), The method using iTopia (iTopia Epitope Discovery System Assay, Beckman Coulter) described in Shin et al., PNAS, Nov 2007; 104: 19073-19078, etc., which uses only HLA fixed to a solid phase and Observe the HLA antibody that specifically binds when the polypeptide binds.

The results of the above partial peptides of the polypeptide represented by SEQ ID No. 1 and SEQ ID No. 2 were discussed, as epitope candidate peptides, represented by SEQ ID NOs: 3, 4, 5, and 72 Polypeptide, and the polypeptide represented by sequence number 10 have been identified set. Therefore, in a more preferable aspect, the polypeptide of the present invention is a polypeptide represented by SEQ ID NO. 3, SEQ ID No. 4, SEQ ID No. 5, or SEQ ID No. 72. Moreover, in another more preferable aspect, the polypeptide of the present invention is the polypeptide represented by SEQ ID NO: 10.

In another aspect, the polypeptide of the present invention is a partial peptide of BORIS belonging to isoform A or C, or subtype 5 or 6, which has the ability to bind HLA-A11 antigen. Since the HLA-A11 antigen is an HLA belonging to HLA type I, the polypeptide in this aspect preferably has about 8 to 11 amino acid lengths.

After discussing the above-mentioned partial peptides of BORIS with anti-HLA-A11 antigen binding ability, as epitope candidate peptides, polypeptides represented by sequence numbers 60 to 73 were identified. As a result of further research on these polypeptides, high CTL inducibility was confirmed among SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, and SEQ ID No. 72. Therefore, in a more preferred aspect, the polypeptide of the present invention is a polypeptide represented by SEQ ID NO: 65, SEQ ID No. 66, SEQ ID No. 67, or SEQ ID No. 72. Among them, the polypeptide represented by SEQ ID NO: 72 is more preferable because it is a partial polypeptide of the unique sequence of BORIS sf6 (SEQ ID NO 1) as described above. In another and even better aspect, the polypeptide of the present invention is a polypeptide represented by SEQ ID NO: 65, SEQ ID No. 66, or SEQ ID No. 67. These are partial polypeptides represented by SEQ ID NO: 76 Peptides, especially those found in the area of zinc fingers. In the past, the zinc finger area of BORIS is highly similar to CTCF, which is known to be widely expressed in somatic cells. Therefore, it has been considered difficult to obtain BORIS. Specific tumor antigen peptides. Therefore, it is surprising that BORIS-specific tumor antigen peptides derived from the zinc finger region were obtained according to the research of the inventors of the present case.

In another aspect, the polypeptide of the present invention is a partial peptide of BORIS belonging to isoforms A or C, or subtype 5 or 6, which has an anti-HLA-A2 antigen and/or HLA- A24 antigen binding capacity. Since the HLA-A2 antigen and the HLA-A24 antigen are HLAs belonging to HLA type I, the polypeptide in its present form preferably has about 8-11 amino acid lengths.

The results of the above-mentioned partial peptides of BORIS, which have the ability to bind to HLA-A2 antigen and/or HLA-A24 antigen, were discussed as epitope candidate peptides, represented by sequence numbers 3-16 and 47-57. The peptide was identified. As a result of further research on these polypeptides, it was confirmed that proper HLA binding was shown in SEQ ID NO. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 10, SEQ ID No. 47, SEQ ID No. 48, and SEQ ID No. 57 ability. Therefore, in a better aspect, the polypeptide of the present invention is a polypeptide represented by SEQ ID NO. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 10, SEQ ID No. 47, SEQ ID No. 48, and SEQ ID No. 57 . Among them, the polypeptides represented by sequence number 3, sequence number 4, and sequence number 5 are more preferable because they are partial polypeptides of the unique sequence of BORIS sf6 (sequence number 1) as described above. The polypeptide represented by the sequence number 10 is a partial polypeptide of the unique sequence of BORIS sf5 (sequence number 2) as described above, and has a binding to both HLA-A2 antigen and HLA-A24 antigen Ability, so better. In another and even better aspect, the polypeptide of the present invention is a polypeptide represented by SEQ ID NO: 47, SEQ ID No. 48, and SEQ ID No. 57. These are also partial successes of the polypeptide represented by SEQ ID No. 76. Peptide. Furthermore, among these, polypeptides represented by SEQ ID NO: 47 and SEQ ID NO: 57 are more preferable.

Among them, particularly preferred are polypeptides represented by sequence number 4, sequence number 5, sequence number 10, sequence number 47, sequence number 48, and sequence number 57. These polypeptides have been confirmed to induce specific cytotoxic T cells (CTL) based on the research conducted by the inventors of the present case.

Among the polypeptides of this aspect, the polypeptides having the ability to bind to the HLA-A2 antigen are not limited to this, but examples include SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 10, and SEQ ID No. The polypeptide represented by 47 and SEQ ID NO: 57, etc.

Among the polypeptides of this aspect, the polypeptide having the ability to bind to the HLA-A24 antigen is not limited to this, but the polypeptide represented by SEQ ID NO. 3 or SEQ ID NO. 10, etc., can be cited.

In another aspect, the polypeptide of the present invention is a polypeptide in which one or more amino acids are added, deleted, or substituted in the above-mentioned polypeptide. Of course, the polypeptide in this aspect is a polypeptide that has been added, deleted, or substituted with one or more amino acids and has HLA-binding ability.

HLA antigens, especially peptides that bind to HLA type I antigens, are known to have specific amino acids at specific positions, which are called anchor motifs. The replacement of the fixed phantom with another anchor phantom is believed to not lose the HLA binding ability. therefore As additions, deletions, and substitutions in the peptide of the present invention, it is preferable to replace the anchor motif with addition, deletion, and substitution of another anchor motif. For example, among polypeptides with HLA-A11 antigen binding properties, it is known that most of them are arranged at the second position from the N-terminus with any one of Ile, Met, Ser, Thr or Val, and in the 9th or 10th position. Any one of Lys or Arg is arranged at the position, as the above-mentioned preferable additions, deletions, and substitutions, for example, the substitution of Ile at the second position from the N-terminus to Met, Ser, Thr or Val .

That is, as examples of preferable additions, deletions, and substitutions of the present invention, (a) in the HLA-A11 antigen-binding peptide, the peptide at the second position from the N-terminus is set as Ile, Met, Ser, Thr or Val; (b) HLA-A11 antigen-binding peptides, the 9th or 10th peptide from the N-terminus is set to Lys or Arg; (c) HLA -In the A24 antigen-binding peptide, the peptide at the second position from the N-terminus is set to Trp, Phe, Met, or Tyr; (d) in the HLA-A24 antigen-binding peptide, the peptide is from the N-terminus The ninth or tenth peptide is Phe, Leu, Ile or Trp; (e) In the HLA-A2 antigen-binding peptide, the peptide at the second position from the N-terminal is set as Ile, Val, Ala, or Thr; and/or (f) HLA-A2 antigen-binding peptides, with the 9th or 10th peptide from the N-terminus being Val, Leu, Ile, Ala, or Met Wait.

The synthesis of the polypeptide of the present invention can be carried out in accordance with a conventional method used in general peptide chemistry. As the way to learn Methods, including those described in the literature (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; peptide synthesis, Maruzen (stock), 1975; peptide synthesis Fundamentals and Experiments, Maruzen (Stock), 1985; Development of Pharmaceuticals Continued Volume 14 ‧ Peptide Synthesis, Guangchuan Bookstore, 1991; These documents constitute part of this application by reference) and other methods.

The polypeptide of the present invention can be confirmed live by providing it to the CTL induction method described later or the analysis method using human model animals (WO02/47474, Int J. Cancer: 100, 565-570 (2002)). In vivo activity.

Since most cancer cells containing cancer stem cells show isoforms A or C, or BORIS belonging to subtype 5 or 6, the polypeptide of the present invention, as described in this specification, can be used for induction Develop cytotoxic T cells (CTL) that specifically recognize these cells. Therefore, the polypeptide of the present invention is beneficial to the prevention and/or treatment of cancer, etc., and can be used as an effective ingredient of a pharmaceutical composition. In particular, since the inventors of the present case found that sf5 and/or sf6 of the BORIS subtype are specifically expressed in cancer stem cells, the polypeptide of the present invention derived from the amino acid sequence specific to this subtype can It is particularly suitable for use in medical compositions for treating cancer stem cells. In addition, the polypeptide of the present invention can also be used for the prevention and/or treatment of cancer. In addition, the present invention also relates to the use of the polypeptide of the present invention in the manufacture of medicines for the prevention and/or treatment of cancer.

(2) Polynucleotide of the present invention

The polynucleotide of the present invention includes at least one polynucleotide that encodes the aforementioned polypeptide of the present invention. The polynucleotide of the present invention can be any of cDNA, mRNA, cRNA or synthetic DNA. It can be either single-strand or double-strand. Specifically, for example, the code can be described in serial number 3, serial number 4, serial number 5, serial number 10, serial number 47, serial number 48, serial number 57, serial number 65, serial number 66, A polynucleotide composed of the nucleotide sequence of the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 72, and a polynucleotide composed of a nucleotide sequence that encodes in each expression, and the like. The polynucleotide of the present invention, in one aspect, is used to produce the polypeptide of the present invention in a host using genetic recombination technology. In this case, since the frequency of use of amino acid codes varies between hosts, the amino acid code can also be changed in order to match the frequency of use of the production host.

The polynucleotide of the present invention can take any form of single strand and double strand. When the polynucleotide of the present invention is double-stranded, by inserting the polynucleotide of the present invention into the expression vector, a recombinant expression vector for expressing the polypeptide of the present invention can be produced. That is, within the scope of the polynucleotide of the present invention, a recombinant expression vector made by inserting the double-stranded polynucleotide of the present invention into a performance vector is also included.

The polynucleotide of the present invention, as described in this specification, is useful for the prevention and/or treatment of cancer, etc., and can be used as an active ingredient of a pharmaceutical composition. In addition, the polynucleotide of the present invention can also be used for the prevention and/or treatment of cancer By. In addition, the present invention also relates to the use of the polynucleotide of the present invention in the manufacture of medicines for the prevention and/or treatment of cancer.

The expression vector used in the present invention can be used in various types depending on the host or purpose used, and can be appropriately selected by a person with ordinary knowledge in the technical field to which the present invention belongs. As the expression vector that can be used in the present invention, for example, plastids, phage vectors, viral vectors, mucus vectors, fosmid vectors, artificial chromosome vectors (HAC, YAC, BAC, PAC) can be mentioned. Wait. For example, when the host is Escherichia coli, examples of the vector include plastid vectors such as pUC118, pUC119, pBR322, pCR3, and pGATA; and phage vectors such as λ ZAPII and λ gt11. When the host is yeast, examples of the vector include pYES2, pYEUra3, and pYAC4. When the host is an insect cell, pAcSGHisNT-A, pIEx, pBAC, etc. can be cited. When the host is an animal cell, pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV, pRc/CMV and other plastid vectors or retroviral vectors, adenovirus vectors, adeno-associated virus vectors, Viral vectors such as vaccinia vector, Sendai virus vector, lentivirus vector, etc.

The aforementioned vector may also appropriately have factors such as an inducible promoter, a gene encoding a signal sequence, a marker gene for screening, and a terminator. Furthermore, in order to facilitate separation and purification, expression sequences may also be added as a fusion protein fused with thiol redox protein, histidine tag (His-tag), or GST (glutathione sulfur transferase). In this case, an appropriate promoter (lac, tac, trc, trp, CMV, SV40 early promoter, etc.) GST fusion protein vector (pGEX4T, etc.), or vector with Myc, His, etc. tag sequence (pcDNA3.1/Myc-His, etc.), as well as performance and thiol oxidation The vector (pET32a) of the fusion protein fused with reduced protein and histidine tag.

By using the expression vector produced above to transform the host, a transformed cell containing the expression vector can be produced. As the host used here, any cell can be used within the range that does not impair the functions of the polypeptide of the present invention, for example, bacteria such as Escherichia coli, attenuated Salmonella, yeast, and insect cells , Animal cells, etc. Examples of Escherichia coli include HB101 strain, C600 strain, JM109 strain, DH5α strain, AD494(DE3) strain, BL21 strain, etc. of the E. coli K-12 system. As the yeast, Saccharomyces cerevisiae and the like can be cited. Examples of animal cells include L929 cells, BALB/c3T3 cells, C127 cells, CHO cells, COS cells, Vero cells, HeLa cells, and 293-EBNA cells. Examples of insect cells include sf9, Hi5, and S2.

As a method of introducing the expression vector into the host cell, any general introduction method suitable for the aforementioned host cell may be used. Specifically, the calcium phosphate method, the DEAE-dextran method, the electroporation method, the method (lipofection method) using lipids for gene transfer (Lipofectamine, Lipofectin; Gibco-BRL), etc. can be mentioned. After the introduction, by culturing in a general medium containing the selection marker, it is possible to screen out the transformed cells in which the aforementioned expression vector is introduced into the host cell.

The polypeptide of the present invention can be produced by continuously culturing the transformed cells obtained as described above under appropriate conditions. The obtained polypeptide can be further separated and purified by general biochemical purification methods. Here, as the purification means, salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, colloidal filtration chromatography, etc. can be mentioned. Furthermore, when the polypeptide of the present invention is expressed as a fusion protein fused with the aforementioned thiol redox protein, histidine tag, GST, etc., it can be performed by a purification method that utilizes the properties of these fusion proteins or tags. Separation and refinement. In addition, when bacteria such as attenuated Salmonella are used as host cells, the bacteria are used as gene delivery vectors. That is, the host cells, that is, bacteria can also be used directly into the body of the subject.

The polynucleotide encoding the polypeptide of the present invention may be in the form of DNA or RNA. The polynucleotides of the present invention, the amino acid sequence information of the polypeptides of the present invention and the DNA sequence information encoded thereby can be easily manufactured using common methods known in the technical field. Specifically, it can be manufactured by general DNA synthesis or amplification by PCR.

The polynucleotide encoding the polypeptide of the present invention includes the polynucleotide encoding the aforementioned epitope peptide.

(3) CTL inducer using the polypeptide of the present invention as an active ingredient

As mentioned above, the polypeptide of the present invention can be used in a method for inducing CTL against cancer cells, so it can be used as a tumor antigen peptide as a CTL inducer.

That is, by separating the peripheral blood lymphocytes from a human blood sample and adding the polypeptide of the present invention to a test tube (in vitro) for stimulation, it is possible to induce specific recognition of HLA pulsed by the peptide. CTL of antigen-positive cells (J. Immunol., 154, p2257, 1995). In this case, the induction of CTL can be confirmed by the following method. For example, the amount of various cytokines (for example, IFNγ) produced by CTL is measured by, for example, ELISA method, which is a peptide presenting cell for antigen React. It can also be confirmed by a method ( ⁵¹ Cr release analysis method, Int. J. Cancer, 58: p317, 1994) that measures the cytotoxicity of CTL to antigen peptides labeled with ⁵¹ Cr.

Furthermore, CTL clones can also be established by the method described in Int. J. Cancer, 39, 390-396, 1987, N. Eng. J. Med, 333, 1038-1044, 1995, etc.

The CTL induced by the polypeptide of the present invention has a toxic effect on the cells presenting the polypeptide of the present invention as an antigen or the ability to produce lymphoids. Since the polypeptide of the present invention is a tumor antigen peptide as described above, it can exert an anti-tumor effect through these functions, preferably an anti-cancer effect. Therefore, the polypeptide of the present invention and the CTL induced therefrom can be used as effective ingredients of medicines or pharmaceutical compositions for the prevention and/or treatment of cancer.

If a CTL inducer containing the polypeptide of the present invention as an effective ingredient is administered to a cancer patient, the polypeptide of the present invention is displayed on the HLA antigen of the antigen-presenting cell, which is effective for the HLA antigen and the displayed peptide. The specific CTL that binds to the complex proliferates and can destroy cancer cells. As a result, cancer can be prevented and/or treated. Therefore, the CTL inducer using the polypeptide of the present invention as an active ingredient is preferably used for subjects positive for HLA-A02 antigen, HLA-A11 antigen, and/or HLA-A24 antigen. Among them, it is even better to treat patients suffering from cancers that express isoforms A or C, or belong to BORIS subtype 5 or 6, and even better to treat cancers that are BORIS sf5 and/or sf6 positive Object to use. Examples of isoforms A or C, or BORIS-positive cancers belonging to subtype 5 or 6, include cancers (tumors) such as cervical cancer, ovarian cancer, uterine cancer, breast cancer, colorectal cancer, lung cancer, and melanoma. The CTL inducer of the present invention can be used for the prevention and/or treatment of these cancers. Particularly, it can be preferably used for the prevention and/or treatment of cancer in female organs such as cervical cancer, ovarian cancer, and uterine cancer, and lung cancer.

In this “prevention” of cancer, not only does it prevent the patient from developing cancer, but also includes cancer treatments such as recurrence prevention, surgery, radiation therapy, or drug therapy in patients who have undergone surgery to remove the primary tumor. Metastasis of completely removed tumors, etc. In addition, in the "treatment" of cancer, it is not only the cure and symptom improvement of the cancer that shrinks the cancer, but also the prevention of cancer cell proliferation, tumor expansion, or prevention of cancer cell metastasis originating from the primary lesion.

The CTL inducer using the polypeptide of the present invention as an active ingredient is for BORIS positive cancers suffering from isoforms A or C, or subtype 5 or 6, preferably BORIS sf5 and/or sf6 positive cancers HLA-A02, HLA-A11 or HLA-A24 positive cancer patients are particularly effective. Specifically, it can be used for the prevention or treatment of cancers (tumors) such as cervical cancer, ovarian cancer, uterine cancer, breast cancer, colorectal cancer, lung cancer, and melanoma. It can be preferably used for the prevention and/or treatment of cancers in female organs such as cervical cancer, ovarian cancer, and uterine cancer.

The dosage form of the CTL inducer using the polypeptide of the present invention as an active ingredient is not particularly limited, but examples include oil emulsions (emulsified preparations), polymer nanoparticles, liposome preparations, and binding to several diameters. the beads μ m particulate formulation, the formulation of lipid binding, microsphere preparations, microcapsule preparations.

The CTL inducer that uses the polypeptide of the present invention as an active ingredient can be administered with a carrier acceptable as a medicine, for example, can be mixed with a suitable adjuvant for administration, in order to effectively establish cellular immunity, or And used to vote.

As the adjuvant, conventional adjuvants in this technical field can be used. Specifically, for example, as the gel formula, aluminum hydroxide, aluminum phosphate, and calcium phosphate can be cited; as the bacterial formula, CpG, mono Monophosphoryl lipid A (MPL), cholera toxin, Escherichia coli heat-labile enterotoxin, pertussis toxin and Muramyl dipeptide (MDP), etc.; as an oil emulsion type (Emulsified preparations) include incomplete Freund’s adjuvant, MF59 and SAF; examples of polymer nanoparticle formula include immunostimulatory complex (Immunostimulatory complex; ISCOMs), liposomes, and biodegradable microspheres ( Biodegradable microsphere) and QS-21 derived from saponin; as synthetic formulas, nonionic block copolymers, Muramyl peptide analogues, polyphosphazenes and synthetic polynucleotides can be mentioned. ; Examples of cytokines include IFN-α, IFN-β, IFNγ, IL-2, and IL-12.

As the administration method, any conventionally known administration methods such as intradermal administration, subcutaneous administration, intramuscular administration, and intravenous administration can be cited. The dosage of the polypeptide of the present invention in the preparation can be appropriately adjusted according to the disease, the patient's age, weight, etc., for the purpose of treatment, but it is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, and more preferably 0.1mg~10mg, it is better to administer this once within several days to several months.

As described above, by using the CTL inducer containing the polypeptide of the present invention as an active ingredient, it is possible to effectively treat isoforms A or C, or BORIS-positive cancers belonging to subtype 5 or 6. Therefore, in one aspect, the present invention includes a pharmaceutical composition containing the CTL inducer as an active ingredient, preferably a composition for cancer stem cell treatment, or a composition for cancer prevention and/or treatment.

(4) CTL inducer using the polynucleotide of the present invention as an effective ingredient

The cell expressing the polynucleotide of the present invention becomes a cell expressing the polypeptide of the present invention as an antigen, and therefore has the characteristic of being recognized by T cells through T cell receptors. Therefore, the polynucleotide of the present invention can also be an inducer of CTL. The induced CTL, like the CTL induced by the polypeptide of the present invention, can exert an anti-tumor effect through cytotoxicity or the production of lymphoid, preferably an anti-cancer effect. Therefore, the polynucleotide of the present invention can be used as an effective ingredient of a medicine or a pharmaceutical composition for the treatment or prevention of cancer. The CTL inducer containing the polynucleotide of the present invention as an active ingredient can treat and/or prevent cancer by, for example, administering the polynucleotide of the present invention to a cancer patient to express it.

For example, if the polynucleotide of the present invention inserted into the expression vector is administered to a cancer patient by the following method, a large amount of tumor antigen peptide is expressed in the antigen presenting cell. After that, the generated tumor antigen peptide and HLA-A02 antigen, HLA-A11 antigen, or HLA-A24 antigen are combined to form a complex, and the complex is displayed at a high density on the surface of antigen-presenting cells, cancer-specific CTL efficiently proliferate in the body and destroy cancer cells. Proceed as described above to achieve cancer treatment or prevention.

The CTL inducer using the polynucleotide of the present invention as an active ingredient is preferably used in a subject that is positive for HLA-A02 antigen, HLA-A11 antigen, and/or HLA-A24 antigen. Among them, it is more preferable for a subject suffering from a cancer that exhibits isoforms A or C, or a BORIS subtype 5 or 6, and even more preferably for a subject who has suffered from BORIS sf5 and/ Or use it for sf6-positive cancer subjects. Examples of isoforms A or C, or BORIS-positive cancers belonging to subtype 5 or 6, include cervical cancer, ovarian cancer, uterine cancer, breast cancer, colorectal cancer, lung cancer, melanoma and other cancers (tumor ) Etc. The CTL inducer of the present invention can be used for the prevention or treatment of these cancers. It can be preferably used for the prevention and/or treatment of cancers in female organs such as cervical cancer, ovarian cancer, and uterine cancer, and lung cancer.

Examples of methods implemented by viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, Sendai virus, lentivirus, pox virus, polio virus, Sindbis virus ( sindbis virus) or other DNA virus or RNA virus inserting the DNA of the present invention into the method. Among these, methods using retrovirus, adenovirus, adeno-associated virus, vaccinia virus, etc. are particularly preferred.

Other methods include the method of directly administering expression plastids into the muscle (DNA vaccine method), liposome method, lipofectin method, microinjection method, calcium phosphate method, electroporation Methods, etc., particularly preferred are DNA vaccine method and liposome method. In addition, a bacterial vector method can also be used, which involves introducing expression plastids into attenuated bacteria such as Salmonella, and administering the bacteria to express the polypeptide of the present invention.

When administering the polynucleotide of the present invention, an appropriate administration route and administration form according to the disease, symptom, etc. of the treatment purpose can be appropriately selected for administration. For example, it can be administered in a form that can be injected into a vein, artery, subcutaneously, intracutaneously, intramuscularly, or the like. In the case of administration, for example, liquid preparations can be obtained in the form of preparations, but generally they can be used as containing active ingredients, that is, The injection of the polynucleotide of the present invention, etc., may be added with a pharmaceutically acceptable carrier (carrier) as necessary. In addition, in liposomes or membrane fusion liposomes (Sendai virus (HVJ)-liposomes, etc.) containing the polynucleotide of the present invention, it can be used as a suspending agent, a refrigerant, a centrifugal separation concentrated refrigerant, etc. The form of liposome preparations.

The content of the polynucleotide of the present invention in the preparation can be appropriately adjusted according to the disease to be treated, the patient's age, weight, etc., but for example, the content of the polynucleotide is 0.0001 mg to 100 mg, preferably 0.001 mg-10 mg of the polynucleotide of the present invention is administered once within several days to several months.

Anyone who has general knowledge in the technical field of the invention can appropriately select suitable cells, vectors, administration methods, administration forms, and dosages.

(5) Antigen presenting cell of the present invention

The aforementioned polypeptide or polynucleotide of the present invention can be used in the treatment of cancer patients, for example, in vitro as described below. That is, by contacting any one of the polypeptide or polynucleotide of the present invention with cells having antigen-presenting ability, antigen-presenting cells can be produced. Although the preparation of antigen-presenting cells may be performed in vitro or in vivo, it is preferably performed in vitro. Specifically, the present invention provides an antigen-presenting cell and a method for manufacturing the same. The antigen-presenting cell is preferably obtained by making cells with antigen-presenting ability isolated from cancer patients, and the present invention Any one of the polypeptide or polynucleotide of the invention is contacted in vitro, and on the cell surface of the cell, for example, HLA-A02 antigen or HLA-A11 antigen, HLA- A24 antigen, etc. and a complex of the polypeptide of the present invention.

The antigen-presenting cell of the present invention includes, for example, (1) the antigen-presenting cell and the CTL epitope peptide are mixed for 30 minutes to 1 hour in an appropriate culture medium to stimulate antigen presentation Cells, (2) using nucleic acid encoding CTL epitope peptides, using gene transfer and other methods to present CTL epitope peptides on antigen presenting cells, (3) artificially made cells with antigen presenting ability Artificial antigen presenting cells, etc.

The so-called "cells with antigen-presenting ability" herein, as long as they are MHC capable of presenting the polypeptide of the present invention, preferably HLA-A02 antigen, HLA-A11 antigen and/or HLA-A24 antigen are expressed on the cell surface The above cells are not particularly limited, but among these, professional antigen-presenting cells are preferred, and dendritic cells considered to be high in antigen-presenting ability are more preferred. Artificial antigen-presenting cells with antigen-presenting capability can be manufactured by the following methods, such as immobilizing HLA and CTL epitope peptides and β 2 on lipid bilayers or plastic or latex beads. -Microglobulin complex of 3, and immobilized costimulatory molecules such as CD80, CD83, and CD86 that can stimulate CTL, or immobilized an antibody, etc., which coordinate with the T cells that bind the costimulatory molecule CD28 played a competitive role.

In addition, as a substance added to prepare the antigen-presenting cell of the present invention from the aforementioned cells having antigen-presenting ability, any one of the polypeptide or polynucleotide of the present invention may be used.

The antigen-presenting cells of the present invention, for example, by isolating cells with antigen-presenting ability from cancer patients, and stimulating the cells in vitro with the polypeptide of the present invention to make HLA-A02 antigen and HLA -A11 antigen and/or HLA-A24 antigen and a complex of the polypeptide of the present invention are presented. When dendritic cells are used, for example, lymphocytes are separated from the peripheral blood of cancer patients by the ficoll method, and then non-adherent cells are removed, and adherent cells are present in GM-CSF and IL-4 The antigen-presenting cells of the present invention can be prepared by culturing the dendritic cells under the condition of inducing dendritic cells, and culturing the dendritic cells together with the polypeptide of the present invention for stimulation.

In addition, when the antigen-presenting cell of the present invention is prepared by introducing the polynucleotide of the present invention into the aforementioned cells having antigen-presenting ability, the polynucleotide may be in the form of DNA or RNA. form. The method of introducing polynucleotides to prepare antigen-presenting cells is known in this technical field, and those with ordinary knowledge in the technical field to which the present invention belongs can appropriately select them.

The aforementioned antigen-presenting cells can be used as effective ingredients of CTL inducers. In order to stably maintain the antigen-presenting cells, the CTL inducer containing the antigen-presenting cells as an active ingredient preferably contains physiological saline, phosphate-buffered physiological saline (PBS), culture medium, and the like. Examples of the administration method include intravenous administration, subcutaneous administration, and intradermal administration. By The CTL inducer containing such antigen-presenting cells as an active ingredient is returned to the patient's body. In the body of a patient suffering from isoform A or C, or BORIS-positive cancer belonging to subtype 5 or 6, The cancer cell-specific CTLs that express the polypeptide of the present invention for antigen presentation are efficiently induced, and as a result, it is possible to treat the isoforms A or C that express the polypeptide of the present invention for antigen presentation, or BORIS positive cancer of subtype 5 or 6.

(6) Cytotoxic T cell (CTL) of the present invention

The polypeptide and polynucleotide of the present invention can be used as described below in the treatment of cancer patients. That is, by contacting any one of the polypeptide and polynucleotide of the present invention with peripheral blood lymphocytes, CTLs can be induced, and in particular, CTLs can be induced to specifically recognize and exhibit isoforms A or C. , Or CTL of cells belonging to the BORIS gene of subtype 5 or 6. In other words, the present invention provides a CTL and a method of inducing the same. The CTL is obtained by contacting peripheral blood lymphocytes derived from cancer patients with any one of the polypeptide or polynucleotide of the present invention Induced. This method can be performed in vitro or in vivo, but it is preferably performed in vitro.

Specific examples of methods for inducing CTL of the present invention include the following methods. Firstly, use the polypeptide of the present invention to directly stimulate PBMC (peripheral blood mononuclear cell; peripheral blood mononuclear cell) or T cell, or use the antigen-presenting cell stimulated by the peptide, the antigen-presenting cell introduced by gene, or artificial Artificial antigen presenting cells with antigen presenting ability are produced for stimulation. The CTL induced by the stimulation was cultured in a 5% CO ₂ thermostat at 37°C for 7-10 days. The stimulation by CTL epitope peptides and IL-2, or by antigen-presenting cells and IL-2 is repeated once a week to ensure the necessary number of CTL cells.

For example, in melanoma, a large number of infiltrating T cells in the patient's tumor are cultured in vitro, and then the cells are returned to the patient by indirect immunotherapy to see the therapeutic effect. In mouse melanoma, the tumor antigen peptide TRP-2 is stimulated by spleen cells in vitro to proliferate CTL specific for the tumor antigen peptide, and the CTL is administered In mice transplanted with melanoma, metastasis inhibition can be seen. This is based on the result of in vitro proliferation of CTL that specifically recognizes the complex of the MHC of the antigen-presenting cell and the tumor antigen peptide. Therefore, after the polypeptide or polynucleotide of the present invention is used to stimulate the peripheral blood lymphocytes of the patient in vitro to increase tumor-specific CTL, the treatment method of returning the CTL to the patient is considered to be useful.

The CTL can be used as an active ingredient of a therapeutic or preventive agent for cancer. The therapeutic or preventive agent preferably contains physiological saline, phosphate buffered physiological saline (PBS), culture medium, etc., in order to stably maintain CTL. Examples of the administration method include intravenous administration, subcutaneous administration, and intradermal administration. A treatment or preventive agent for cancer that contains such CTL as an active ingredient returns to the patient's body, and is suffering from the isoform A or C of the present invention, or a BORIS positive cancer belonging to subtype 5 or 6 The toxic effect of cancer cells in the body of patients by CTL is promoted, thereby destroying cancer cells, and can treat cancer.

(7) HLA-multimer of the present invention

The so-called HLA-tetramer refers to the biotinylation of a complex (HLA-monomer) formed by the assembly of HLA and β2 microglobulin and a peptide (antigenic peptide) to bind it to avidin It becomes a tetramer (Science 279: 2103-2106 (1998), Science 274: 94-96 (1996)), which is described in, for example, US Patent No. 5,635,363, French Patent Application Publication No. FR9911133, and US Patent No. 5,723,584, US Patent No. 5,874,239, US Patent No. 5,932,433, US Patent No. 6,265,552, Japanese Patent Registration No. 4976294, etc. At present, HLA-tetramers containing various antigen peptides are being produced, and HLA-tetramers containing the polypeptide of the present invention and HLA-A02, HLA-A11 or HLA-A24 can be easily produced. In addition, HLA-dimer and HLA-pentamer are also based on the same principle. In these multimers, the aforementioned HLA monomer is dimerized and pentamerized, respectively. Therefore, containing the polypeptide of the present invention, that is, having isoforms A or C, or the HLA binding ability of BORIS belonging to subtype 5 or 6, especially partial peptides with HLA type I binding ability, HLA monomers and HLA multimers, especially HLA-tetramers, and HLA-A02, HLA-A11 or HLA-A24 are also aspects of the present invention.

Specifically, for example, an HLA-tetramer containing the polypeptide of the present invention and HLA-A02, HLA-A11, or HLA-A24 can be cited. The HLA-tetramer is preferably fluorescently labeled in order to be able to easily select or detect the bound CTL by using conventional detection methods such as flow cytometry and fluorescence microscopy. Specifically, for example, an HLA-tetramer labeled with phycoerythrin (PE), fluorescent isothiocyanate (FITC), dinofoxanthin-chlorophyll-protein (PerCP), etc. can be mentioned.

Examples of the HLA-tetramer manufacturing method include those described in US Patent No. 5,635,363, French Patent Application No. FR9911133, Science 279: 2103-2106 (1998), Science 274: 94-96 (1996), etc. A documenter can select an appropriate method as long as he has general knowledge in the technical field to which the invention belongs. As a production example, if it is briefly described, it is as follows.

First, the expression vector of HLA-A24, HLA-A11 or HLA-A02 and β 2 microglobulin expression vector are introduced into E. coli or mammalian cells that can express proteins. Here, it is preferable to use Escherichia coli (for example, BL21). The obtained monomer HLA-A24, HLA-A11 or HLA-A02 complex and the polypeptide of the present invention are mixed to form a soluble HLA-peptide complex. Secondly, the C-terminal sequence of HLA-A02, HLA-A11 or HLA-A24 in the HLA-peptide complex is biotinylated by BirA enzyme. By mixing the biotinylated HLA-peptide complex and fluorescence-labeled avidin at a molar ratio of 4:1, an HLA-tetramer can be prepared. In addition, in each of the foregoing stages, it is preferable to perform protein purification by colloidal filtration or the like.

By using the HLA-tetramer (or monomer) of the present invention, the tumor-specific CTL of the present invention can be detected and refined. As a method of generating CTL, the following methods are mentioned, for example.

(i) Allow PBMC to react with the HLA-tetramer of the present invention at an appropriate concentration. The CTL bound to the HLA-tetramer of the present invention is stained by the labeled pigment, and only the stained CTL is separated using a cell sorter, microscope, etc. The CTLs isolated in this way are passed through T cell stimulating agents such as anti-CD3 antibodies, PHA, IL-2, etc., or X-ray irradiation or mitomycin treatment, etc., and pass through antigen-presenting cells that have lost their proliferation ability. Stimulate proliferation and ensure the necessary number of cells.

(ii) Immobilize the HLA-monomer and/or tetramer of the present invention on a sterile dish, etc., and then culture PBMC on the immobilized dish. In order to separate the CTL bound to the HLA-monomer and/or tetramer of the present invention that has been solid-phased on the disc, after washing away other unbound and floating cells, only the CTL remaining on the disc The specific CTL is suspended in a new medium. The CTLs isolated in this way are stimulated by anti-CD3 antibodies, PHA, IL-2 and other T cell stimulating agents, or X-ray irradiation or mitomycin treatment, etc., and are stimulated by antigen-presenting cells that have lost their proliferation ability Proliferate to ensure the necessary number of cells.

(iii) Coordination of the HLA-monomer and/or tetramer of the present invention with costimulatory molecules such as CD80, CD83, and CD86, or with an antibody, etc., on T cells bound to the costimulatory molecule CD28 played a competitive role, solid phased on a sterile plate, etc., and then cultured PBMC on the solid phased plate. After 2 days, IL-2 was added to the medium, and cultured in a 5% CO ₂ constant temperature bath at 37°C for 7 to 10 days. The cultured cells were recovered and continued to be cultured on a new immobilized plate. By repeating this operation to ensure the necessary cell number of CTL.

In addition, by using it in combination with antibodies against cell surface proteins (CD62L, CCR7 or CD45RA, etc.), it is possible to investigate the differentiation stage of CTL (Seder RA, Ahmed R., Nat Immunol., 2003; 4:835-842) . Or, by combining with intracellular cytokinin staining method, it can also be used to evaluate the function of CTL. Therefore, as long as the CTL epitope peptide is identified and the HLA-tetramer is produced, the CTL induced by the epitope peptide can be quantified and qualitatively obtained, and the epitope peptide can be obtained. It can be a huge contribution to the diagnosis information of diseases related to the source protein.

(8) Tumor detection method (examination method, diagnosis method)

The present invention also provides a tumor detection method (examination method, diagnosis method) using the aforementioned HLA-tetramer of the present invention.

The detection method (diagnostic method) of the present invention using the HLA-tetramer of the present invention typically involves collecting blood from a subject, or using biopsies to collect test tissues suspected of being tumors The HLA-tetramer of the present invention is used to detect and determine the tumor antigen peptides contained in the specimen that can be identified from isoforms A or C, or belong to subtype 5 or 6 of BORIS And HLA antigen complex The amount of CTL to detect, check or diagnose cervical cancer, ovarian cancer, uterine cancer, breast cancer, colorectal cancer, lung cancer, melanoma equivalent isoform A or C, or belong to subtype 5 or 6. BORIS positive cancer (tumor) or its degree. In particular, it is possible to detect, examine or diagnose whether or not suffering from cervical cancer, ovarian cancer, uterine cancer, and other cancers and lung cancers that are unique to women, or their extent.

Using the HLA-tetramer of the present invention, CTL can be quantified. The CTL is for BORIS in a biological sample collected by a subject, especially for isoforms A or C, or belongs to subtype 5 or BORIS of 6 is specific. Quantification can be carried out as follows, for example. The peripheral blood or PBMC collected from the subject is reacted with an appropriate concentration of HLA-tetramer. Since the CTL bound to HLA-tetramer is stained by the labeled pigment, it is counted using a flow cytometer, microscope, etc. When reacting with HLA-tetramer reagent, it can also be judged at the same time by using anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, etc. which are labeled with a pigment different from HLA-tetramer reagent T cell subsets of BORIS-specific CTL.

The detection (inspection, diagnosis) method of the present invention can also detect (inspection, diagnosis), for example, in a patient with a tumor, in the case of administering a therapeutic drug to improve the tumor, whether the tumor is improved or improved degree. In addition, the detection (examination, diagnosis) method of the present invention can also be used in the selection of cancer patients, and the cancer can be effectively applied to medicines that use the polypeptide or polynucleotide of the present invention as an effective ingredient, or can be used by The prediction and judgment of the therapeutic effect of the medicine.

The specific aspect of the detection (inspection) method of the present invention using the HLA-tetramer of the present invention includes the following steps (a) and (b), and optionally including (c): (a) let by The step of contacting the biological sample obtained by the subject with the HLA-tetramer of the present invention; (b) the step of measuring the amount of CTL by using the amount of cells bound by the above-mentioned HLA-tetramer as an index, and the CTL will identify the source From the complex of the tumor antigen peptide of BORIS sf5 or sf6 and the HLA antigen in the biological sample; (c) based on the result of (b), the step of judging cancer.

A specific aspect of the diagnostic method of the present invention using the HLA-tetramer of the present invention includes the steps (a), (b) and (c) above.

One aspect of the detection method (inspection method, diagnosis method) of the present invention using the HLA-tetramer of the present invention is by detecting the polypeptide-specific CTL of the present invention in a biological sample and measuring its amount. Implement. For example, by making the HLA-tetramer of the present invention, and using this HLA-tetramer, the antigen peptide-specific CTLs in peripheral blood lymphocytes of patients suspected of having cancer can be quantified by flow cytometry. get on.

The prediction, judgment, judgment or diagnosis of the presence or absence of a tumor, for example, can be performed by measuring the amount of the polypeptide-specific CTL of the present invention in the blood of a subject or a test tissue suspected of a tumor, or by measuring the present invention The amount of polypeptide cells is carried out. At this time, according to the situation, the BORIS gene or isoform A or C in normal corresponding tissues, or the mRNA expression level of BORIS belonging to subtype 5 or 6, can be determined by the present invention. The polypeptide level or CTL level is used as a reference value, and the reference value is compared with the aforementioned level in the sample obtained from the subject to determine the difference between the two.

Here, the comparison of the aforementioned level between the test tissue of the subject and the normal corresponding tissue can be performed by measuring the subject's biological sample and the normal subject's biological sample as objects. Without parallelization, the polypeptide-specific of the present invention can be obtained by measuring normal tissues using plural numbers (at least 2, preferably 3 or more, and more preferably 5 or more) under equal measurement conditions. The average or statistical median value of sexual CTL is used as the normal value, that is, the benchmark value, for comparison.

In addition, in subjects who have been administered the polypeptide or polynucleotide of the present invention, by measuring the amount of the polypeptide-specific CTL of the present invention, it can also be determined whether CTL has actually been induced . For example, the amount of the polypeptide-specific CTL of the present invention in the tissue of the subject is compared with the level of a normal person, for example, a case where it is more than 2 times, preferably more than 3 times as an indicator It can be determined that the treatment implemented by the polypeptide or polynucleotide of the present invention is effective.

(9) Cancer prevention and/or treatment methods

The present invention also relates to a method for preventing and/or treating cancer in a subject, the method comprising effective ingredients selected from the group consisting of polypeptides, polynucleotides, CTLs, and antigen-presenting cells of the present invention The amount is administered to the step where the active ingredient is deemed necessary.

The "subject" in the present invention means any biological individual, preferably an animal, more preferably a mammal, and even more preferably a human individual. In the present invention, although the subject may be healthy or suffering from certain diseases, in the case of seeking prevention and/or treatment of cancer, it typically means suffering from cancer, or at risk of suffering Object. In one aspect of the present invention, the subject is HLA-A02 positive, HLA-A11 positive, and/or HLA-A24 positive. In one aspect of the present invention, the subject is suffering from isoforms A or C, or a BORIS positive cancer of subtype 5 or 6, or is at risk of suffering. In one aspect of the present invention, the subject is HLA-A02 positive, HLA-A11 and/or HLA-A24 positive, and suffers from isoforms A or C, or is a BORIS positive cancer of subtype 5 or 6 , Or there is a risk of suffering.

As the polypeptide, polynucleotide, CTL, and antigen presenting cell of the present invention used in the preventive/therapeutic method of the present invention, any of those described in this specification can be cited. The effective amount in the present invention is, for example, an amount that alleviates the symptoms of cancer, or delays or stops its progression, and is preferably an amount that inhibits cancer or cures cancer. Also, it is preferably an amount that does not produce adverse effects, and the adverse effects exceed the benefits resulting from the investment. This amount can be appropriately determined by an in vitro test using cultured cells, etc., or a test in model animals such as mice and rats. Such a test method is for those with ordinary knowledge in the technical field of the invention. Well known. The specific dosage of the active ingredient can take into account various conditions regarding the subject that the active ingredient is necessary, such as the severity of symptoms, the general health status of the subject, age, The weight, sex of the subject, timing and frequency of eating, administration, concurrent medicine, response to treatment, dosage form, and compliance with treatment are determined.

As a specific dosage, for example, in the case of the polypeptide of the present invention, it is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, and more preferably 0.1 mg to 10 mg. The polypeptide can be used for several days to several months. It is better to invest within 1 time. In addition, in the case of the polynucleotide of the present invention, it is usually 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg, and it is better to administer the polynucleotide once within several days to several months. In addition, as the administration method, any conventionally known appropriate administration method such as intradermal administration, subcutaneous administration, intramuscular administration, and intravenous administration can be used.

One aspect of the prevention/treatment method of the present invention further comprises screening HLA-A02 positive, HLA-A11 positive, and/or HLA-A24 positive subjects as prevention/treatment targets before the step of administration step. This aspect of the present invention may further include a step of determining the HLA type of the object before the above-mentioned screening step. The determination of the HLA type of the object can be made by any conventional method. In addition, one aspect of the prevention/treatment method of the present invention is that before the step of administration, it further includes screening for subjects with isoforms A or C, or BORIS-positive cancers of subtype 5 or 6 Steps as the object of prevention/treatment. This aspect of the present invention may further include a step of detecting isoforms A or C in the subject, or BORIS positive cancer belonging to subtype 5 or 6, before the above-mentioned screening step. The detection of isoforms A or C in the subject, or BORIS positive cancer belonging to subtype 5 or 6, can use the tumor detection described in (8) above method. One aspect of the prevention/treatment method of the present invention is that before the step of administration, it further includes screening for HLA-A02 positive, HLA-A11 positive, and/or HLA-A24 positive, and has isoform A Or C, or a procedure for a subject with BORIS positive cancer of subtype 5 or 6 as a subject of prevention/treatment. This aspect of the present invention may further include the step of determining the HLA type of the subject and detecting the isoform A or C in the subject, or the BORIS positive of subtype 5 or 6 before the above-mentioned screening step. Steps to cancer.

All patents, applications and other publications mentioned in this specification are used in this specification by referring to the entirety.

(10) Antibody of the present invention

The present invention also provides an antibody that specifically binds to isoforms A or C, or is at least a part of BORIS belonging to subtype 5 or 6, preferably with sequence number 1, sequence number 2, or sequence number At least a part of the polypeptide represented by 76 is more preferably at least a part of the polypeptide represented by SEQ ID NO: 1 or 2. Therefore, the antibody of the present invention is preferably capable of specifically identifying BORIS belonging to subtype 5 or 6. The antibody may be a multi-strain antibody or a monoclonal antibody, but it is preferably a monoclonal antibody.

In addition, the antibody of the present invention also includes functional fragments of antibodies such as Fab, Fab', F(ab')2, Fv, scFv, dsFv, Diabody, and sc(Fv)2. Furthermore, multimers of these functional fragments (for example, dimers, trimers, tetramers, polymers) are also included in the antibody of the present invention.

The production of the antibody of the present invention can be carried out in accordance with a method known in this technical field. For example, all or a part of the polypeptide represented by sequence number 1 or 2 can be used as an immunogen to immunize rabbits and the like, and antibodies can be obtained by purifying the serum.

The antibody of the present invention is an antibody that can specifically bind to isoforms A or C, or BORIS belonging to subtype 5 or 6, and can be detected to show isoforms A or C, or belong to subtypes 5 or 6 BORIS cells, or detect isoforms A or C, or belong to subtype 5 or 6 BORIS itself. Therefore, in one aspect of the present invention, a kit for detection and/or purification of BORIS protein containing the antibody of the present invention is provided.

The kit of the present invention is not particularly limited as long as it contains the antibody of the present invention. It includes all kits known in the technical field. Although not limited to this, it can be used for ELISA method and western ink dispensing. Methods, chromatography, immunostaining and other kits.

In addition to the antibody of the present invention, the kit of the present invention may further include one or more arbitrary constituent parts suitable for the use of the kit. Although the constituent parts are not limited to this, they may include For example, secondary antibodies, color reagents, solvents, buffers, positive controls, negative controls, reaction vessels, pretreatment reagents, blocking reagents, slides, coverslips, Instructions for use, etc.

(10) Other

The present invention is based on the knowledge that BORIS proteins, especially BORIS sf5 and/or sf6, are highly expressed in cells having stem cell properties in cervical cancer or ovarian cancer. Therefore, various technologies based on this knowledge are included in the present invention.

The present invention, in one aspect, relates to antibodies that specifically recognize BORIS sf5 or sf6. This antibody can be produced using a method known in the technical field in order to recognize a polypeptide having the amino acid sequence described in SEQ ID NO: 1 or 2 or a part thereof as an epitope. By using the antibody, for example, the expression level of BORIS sf5 and/or sf6 in specific tissues and/or cells can be detected, and thereby, the presence of cancer stem cells or tumors in the tissues or subjects can be determined. In addition, by using the antibody to inhibit the function of BORIS sf5 and/or sf6, which affects the stem cell properties of cancer stem cells, treatment of cancer stem cells and cancer prevention and/or treatment can be performed.

Furthermore, in another aspect of the present invention, it relates to a polynucleotide having a sequence complementary to the BORIS gene. As mentioned above, it is believed that BORIS genes, especially their specific subtypes, will be particularly powerful in cancer stem cells, and affect stem cell properties. Therefore, it is believed that by inhibiting the expression of the BORIS gene, the stem cell properties of cancer stem cells can be suppressed. Therefore, in a preferred aspect, the polynucleotide of the present invention can be used as an inhibitory nucleic acid, especially as an siRNA. In addition, the polynucleotide of the present invention can also be used as a primer or probe for detecting BORIS sf5 and/or sf6 DNA or mRNA in a sample.

In yet another aspect of the present invention, it relates to a method for detecting cancer stem cells, which comprises detecting BORIS sf5 in a sample obtained from a subject And/or the level of mRNA and/or polypeptide of sf6; and, the level of BORIS sf5 and/or sf6 mRNA and/or polypeptide in normal tissues and/or cells is used as the reference value, and the aforementioned detection Compare the level with the aforementioned benchmark. As a method for detecting the level of mRNA and/or polypeptide, a method known in the technical field can be used. As a method of detecting the level of mRNA, for example, RT-PCR, DNA microarray, and northern ink spotting can be cited. In this method of detecting the level of mRNA, the primers and/or probes of BORIS sf5 and/or sf6 can be used. Examples of methods for detecting the level of polypeptides include immune tissue staining and western blotting. In this method for detecting the level of polypeptide, the above-mentioned BORIS sf5 and/or sf6 specific antibodies can be used.

The present invention, in another aspect, relates to a method for detecting tumors in a subject, which includes detecting the level of BORIS sf5 and/or sf6 mRNA and/or polypeptide in a sample obtained from the subject; and, The level of BORIS sf5 and/or sf6 mRNA and/or polypeptide in normal tissues and/or cells is used as a reference value, and the aforementioned detected level is compared with the aforementioned reference. As a method for detecting the level of mRNA and/or polypeptide, a method known in the technical field can be used. As a method of detecting the level of mRNA, for example, RT-PCR, DNA microarray, and northern ink spotting can be cited. In this method of detecting the level of mRNA, the primers and/or probes of BORIS sf5 and/or sf6 can be used. Examples of methods for detecting the level of polypeptides include immune tissue staining and western blotting. In this method for detecting the level of polypeptide, the above-mentioned BORIS sf5 and/or sf6 specific antibodies can be used. In the present invention, the The method of detecting tumors in different aspects can replace the method of (8) above. Therefore, it can also be used, for example, in the cancer detection step in (9) above.

In yet another aspect of the present invention, it relates to a medical composition for the treatment of cancer stem cell-related cancers. The cancer is a partial peptide containing BORIS protein or its isoforms, which is unique to women. Cancer. BORIS is low in cancer-testis antigens, especially in tissues other than testicles. On the other hand, the findings discovered by the inventors of this case are unique to women in cervical cancer, ovarian cancer, and uterine cancer. Carcinomas in the organs, including those containing stem cell-like cells, are strongly expressed. Therefore, it is expected that particularly excellent effects such as low side effects and high specificity will be exhibited in the treatment of cancers in female organs.

[Example]

Examples are shown below to specifically explain the present invention, but the present invention is not limited to these examples. Unless otherwise specified, the experimental method is to use the method generally used in the technical field, such as the method described in the "Immune Experimental Operation Method" (edited by Uda Shunsuke, Konta Shin, Honshusuke, Hamaoka Toshiyuki).

In addition, the isoforms and subtypes of BORIS used in the following examples are shown in the following table. Unless otherwise stated, the case where the name of the isomer is stated is deemed to mean the specific isoform, and the case where the name of the subtype is stated is deemed to mean any of the isoforms belonging to the subtype , The case of "BORIS" alone is deemed to mean the expression product of the general BORIS gene or the BORIS B0 isoform without specifying the isoform or subtype. Also, in the following examples Here, "peptide name-Tet" means an HLA tetramer combined with the peptide represented by the peptide name.

<Example 1> Isolation and identification of cancer stem cells (1) Separation of spheroid forming cells

It has been reported that in cervical cancer, spheroid formation is one of the markers of cancer stem cells. Then, a spheroid formation analysis method using a low-adhesion petri dish was performed. Various cervical cancer cell lines (CaSki, TCS, MS751, SKG-IIIb, ME-180, and SiHa) were cultured using a porous plate with an ultra-low attachment surface (Ultra Low Attachment 6-well plate, manufactured by Corning). The cells are adhered and cultured using a solution of 2mM EDTA added 0.25% trypsin to peel them, and seed them so that there are ¹⁰³ cells/well in each well. As a medium, a serum-free DMEM/F-12 medium supplemented with 20ng/ml h-EGF (obtained by R&D systems), 10ng/ml b-FGF (obtained by R&D systems), and 1% penicillin/chain For those with tetracycline (obtained from GIBCO), 4μg/ml heparin, and N2 supplement (obtained from WAKO) at a final concentration of 1%, cultured under normal culture conditions for 7 or 14 days, in all cells The formation of spheroids of 100 μm or larger was confirmed in the strain (Figure 1). In the following test, the cell population represented by "sphere" means the cell population separated from the spheroid formed by the same non-adherent culture as in this test. In addition, the cell population represented by "bulk" means a cell population obtained by general attachment culture.

(2) The nature of spheroid-forming cells

In order to confirm that the spheroid-forming cells are cells that exhibit stem cell-like properties, radiation tolerance tests, anticancer drug tolerance tests, and flow cytometric analysis were carried out. After comparing the spheroid group with the massive group, it is more It is confirmed that it is a group that exhibits stem cell-like properties.

(3) Expression analysis of stem cell sex genes

Using SOX2, NANOG, and Oct3/4 as stem cell genes, the expression levels of the aforementioned genes in the clump and sphere populations of CaSki and TCS cell lines were analyzed by quantitative RT-PCR. The PCR machine uses the STEPONE realtime PCR system (manufactured by Applied Biosystems), and the gene performance is detected by the threshold cycle number (Ct). The ΔΔCt method is used to quantify the expression of stem cell genes in the mass population. Quantify the relative expression at 1 time. The primer and probe mixture for SOX2, NANOG, Oct3/4 uses TaqMan gene expression (Applied Biosystems).

The results are shown in Figure 2. It can be seen that no matter which cell line it is in, the stem cell gene is highly expressed in the cells of the spheroid group. This teaches that cancer cells in the sphere group that exhibit stem cell-like properties are concentrated. Using the CaSki cell line that particularly exhibits stem cell genes, the following cDNA microarray analysis was performed.

(4) cDNA microarray

In order to analyze genes that are highly expressed in the sphere group compared to the block group, a cDNA microarray is used. First, a commercially available aminoallyl RNA amplification kit ver2 (high-yield type) (Sigma Aldrich) was used to extract total RNA from each cell in accordance with the instructions attached to the kit. 3 μg of the obtained total RNA was reverse transcribed using a commercially available oligo dT T7 promoter primer and reverse transcriptase to synthesize cDNA. Secondly, T7 RNA polymerase was used to synthesize cRNA, and Cy3 or Cy5 labeled cytidine triphosphate was introduced. With this procedure, Cy5 is used to label samples of spheroid cells. A sample of clumped cells was used as a control cell and labeled with Cy3. NanoDrop (Thermo Scientific Company) was used again to confirm the quality of cRNA. Then it was combined with Cy3 labeled cRNA and Cy5 labeled cRNA, and fragmented in a hybridization cocktail (Sigma Aldrich). The labeled cRNA was hybridized in a 60mer probe oligonucleotide microarray (Panorama Human Micro Array, Sigma Aldrich) and incubated at 50°C for 20 hours. Use Genepix 4000B Microarray Scanner (Axon Instruments) to determine the fluorescence intensity. Using the same method, use Cy3 to label the sample of spheroid cells, and use Cy5 to label the sample of massive cells and perform the experiment again (Dye Swap method).

(5) Selection of candidate proteins for cancer stem cell specific antigen

In the results of the cDNA microarray described above, cancer-testicular antigen (CT antigen) was used as the target, and the expression in the spheroid group was confirmed. The results are shown in the table below.

As a CT antigen specifically expressed in the sphere group, BORIS has been identified. Therefore, it is taught that BORIS can be an effective treatment target for cancer stem cells.

<Example 2> Evaluation of BORIS as a therapeutic target for cancer stem cells (1) Analysis of BORIS performance in normal tissues

As a cDNA gene library of normal tissues, Human Multiple Tissue cDNA Panels I and II (Clontech) were used. PCR is a cDNA mixture containing 0.1~0.5 μl of cDNA, 0.1 μl of Taq DNA polymerase (Qiagen) and 12pmol of primers. After heating at 94°C for 2 minutes, it is then subjected to 30-40 cycles. The cycle is a 15-second dissociation at 94°C, a 30-second bonding at 60°C, and a 30-second extended cycle at 68°C. The primer uses sequence numbers 35 and 36.

The results are shown in Figure 3. It can be seen that BORIS has almost no performance in normal tissues except in the testicles.

(2) Analysis of BORIS performance in each cervical cancer cell line

Using MS751, TCS, CaSki, SKG-IIIb, ME-180 and SiHa as cervical cancer cell lines, cDNA was collected from the clumps and spheres of each cell line using the same method as in Example 1 (4) above, and The expression level of BORIS in each cell line was quantified using the same method as in Example 1 (3) to quantify the relative expression level when the expression in the mass of the TCS cell line was regarded as 1. BORIS primer and probe mixture is used TaqMan gene expression (Applied Biosystems).

The results are shown in Figure 4. In more than half of the cell lines, the expression level of BORIS in the sphere group is greatly increased compared with the block group.

(3) Analysis of BORIS performance in other cancer cell lines

RL95-2 and HEC-1-A as uterine cancer cell lines, and TOV-21G, ES-2, MCAS, Ovcar-3, SMOV-2 and SKOV-3 as ovarian cancer cell lines The expression level of BORIS in the cell is quantified as the relative expression level in which the expression level in the mass of TCS cells is regarded as 1 similarly to (2).

The results are shown in Figure 5. BORIS shows high expression not only in cervical cancer, but also in uterine body cancer and ovarian cancer.

(4) Expression analysis of the isoforms of each subtype of BORIS in cervical cancer cell lines

The expression level of the isoforms of each subtype of BORIS in the cervical cancer cell line was analyzed by RT-PCR in the same manner as (1). Use the primers in the table below as specific primers for each subtype.

[table 3]

The results are shown in Figure 6. Compared with the clumped cells of the CaSki cell line, significant performance can be seen in subtypes (sf) 1 to 4. In the spheroid clumps of cells, sf6, which has almost no performance in the clumps of cells, is strongly expressed . In addition, even in the MS751 cell line, sf1 and sf6 were not observed in the clump cells but were observed in the spheroid cells.

(5) Performance analysis of BORIS subtype variants in ovarian cancer cell lines

The ovarian cancer cell lines TOV21G and SMOV-2 were used instead of the cervical cancer cell lines CaSki and MS751, and the same experiment as the above (4) was performed. The results are shown in Figure 7.

Similar to the results of the above cervical cancer cell lines, even in ovarian cancer cell lines, the specific expression of BORIS sf6 was observed in spheroid cells. In addition, in ovarian cancer cell lines, the performance of not only sf6 but also sf5 is significantly higher than that of clumped cells regardless of the cell line.

(6) Performance analysis of each subtype variant of BORIS in lung cancer cell lines and primary cultured cells established from surgical resection of lung cancer patients

Using cell lines derived from small cell lung cancer, non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma, and primary cultured cells established from surgical resection slices of lung cancer patients, the same experiment as the above (5) was performed. The spheroid-forming cells were prepared using the same procedure as in Example 1 (1) above, except that a medium without N2 supplement and heparin was used. The results are shown in Figure 19-1, Figure 19-2, Figure 19-3, Figure 19-4 and Figure 19-5.

<Example 3> Discussion on BORIS sf6 (1) The production of BORIS isoforms excess expression strain

Using the pMXpuro vector and the Platinum retrovirus expression system containing Plat-A cells, the sf1 isoforms of BORIS, which is the B0 isoforms, and the sf4 isoforms, which are the B3 isoforms, and sf6 isoforms, were produced. The isomers are the retroviral vectors of the B6 isoforms and C7 isoforms, and are introduced into the TCS cell line to produce individual excess expression strains of the BORIS isoforms. Use quantitative PCR to confirm the expression of BORIS isoforms When compared with those who introduced mock vectors separately, it was confirmed that the performance was about 10,000 times higher. By the same method, an excess expressing strain introduced into the SKG-IIIb cell line was also produced.

(2) Sphere formation analysis

Using the same method as in Example 1 (1) above, 1000 cells/well of the excess expressing TCS cell line and the excess expressing SKG-IIIb cell line were seeded, and the spheroid formation analysis was performed by culturing for 2 weeks.

The results are shown in Figure 8 and Figure 9. Regardless of the cell line, significant sphere formation was confirmed in BORIS sf6, especially BORIS B6 isoforms with a large number of expressive strains.

(3) Extraction of candidate BORIS sf6 specific HLA binding epitope

The epitope peptides that bind to HLA type I molecules and are presented by antigens are composed of 8-10 amino acids, the second from the N-terminal side, and the 9th or 10th amino acid The most important amino acid for binding to HLA type I molecules is called the anchor motif. It has been reported that this anchoring phantom will vary depending on the type of HLA type I molecule. For example, the world's most advanced research on epitope peptides that bind to HLA-A2 molecules, the most well-known peptides are composed of 9-10 amino acids, which are located at the second position from the N-terminal. It has leucine on the top, and leucine or valerine on the 9th or 10th position. In addition, as a peptide bound to HLA-A24 molecules, the most well-known peptide is composed of 9-10 amino acids. A peptide having any one of tyrosine, amphetine, methionine or tryptophan at the second position from the N-terminus, and leucine, isoleucine, at the 9th or 10th position Either tryptophan or phenylalanine.

From the specific C-terminal sequence of BORIS sf6 (SEQ ID NO: 1), a peptide with the above-mentioned HLA binding anchor motif structure was extracted. KLLFIGTIKV (KLL peptide: sequence number 4) and LLFIGTIKV (LLF peptide: sequence number 5) as candidate HLA-A2 binding peptides, and SFKKLLFIGTI as candidate HLA-A24 binding peptides (SEQ ID NO. 3) ), synthesized by the usual method.

(4) HLA-A2 binding analysis

The T2 cells were cultured at 26°C overnight. Then wash the cells with PBS, and add the KLL peptide and LLF peptide synthesized in (3) as candidate HLA-A2 binding peptides. The peptide derived from cytomegalovirus is CMV peptide (SEQ ID NO: 37) And the peptide derived from influenza virus, namely Influenza (SEQ ID NO: 38) as a positive control, and the HLA-A24 binding peptide, namely GK-12 peptide (SEQ ID NO: 39) as a negative control, performed at 26°C 3 hours of co-cultivation. After the temperature was set at 37°C and the co-cultivation was carried out for another 3 hours, the supernatant was removed by centrifugation, and the cells were separated. Add HLA-A2 antibody to the separated cells, let stand at 4°C for 1 hour, and then wash with PBS. Add fluorescently labeled anti-mouse IgG+IgM antibody as the secondary antibody. After standing at 4°C for 30 minutes, add 1% formaldehyde to fix the cells. Put the fixed cells in flow cytometry An analyzer (BECTON DIKINSON Company or Beckman Coulter Company) measures the FITC fluorescence intensity.

The results are shown in Figure 10. T2 cells cultured with KLL peptide and LLF peptide showed the same level of fluorescence intensity as CMV peptide and Influenza peptide, and it can be seen that the HLA-A2 molecules were localized on the cell surface to the same extent. . This result teaches that any peptide binds to HLA-A2 and is presented by antigen on the cell surface.

(5) BORIS sf6-specific CTL induction by polypeptide stimulation

Peripheral blood was collected from 2 adults who were known to maintain HLA-A*02:01 health, and centrifuged at 3,000 rpm for 5-10 minutes, and the plasma portion of the supernatant was collected. From the plasma fraction, PBMC was separated by density gradient centrifugation. To each well of a 96-well U-bottom cell culture micro test plate (BECTON DIKINSON), add 10 mL of medium and approximately 3×10 ⁷ PBMCs per plate to the above separated PBMC, and suspend them for culture. It is the Hepes modified RPMI1640 medium (Sigma) with 2-mercaptoethanol (final concentration 55μM), L-glutamic acid (final concentration 2mM), streptomycin (final concentration 100μg/mL) and penicillin G as antibiotics (Final concentration 100U/mL) and 5% plasma components. Among them, BORIS sf6-specific CTL epitope candidate peptides of SEQ ID NO: 4 or SEQ ID NO: 5 were added at a concentration of 10 μg/mL. After 2 days of culture, IL-2 was added at a final concentration of 50 U/mL, and cultured for another 2 weeks.

Culturing the cells for a suitable amount, is added 10 μ L of HLA- PE-labeled tetramer reagent and 20 μ L of CD8-FITC antibody, smoothly mixed, allowed to stand at 4 ℃ 30 minutes. After 1.5 mL of PBS was added and stirred, and centrifuged at 3,000rpm rpm for 5 minutes, the supernatant discarded and the cells were resuspended in 400 μ L of PBS and analyzed using flow cytometry within 24 hours.

The analysis is carried out in two stages. First, the first stage is to collect the cells of the 8-well column in the 96-well U-bottom cell culture micro-test plate as one sample, and confirm whether the BORIS-specific CTL is induced in this sample. For samples in which CTL induction was confirmed at this stage, the second stage analysis was performed. In the second stage, cells were collected individually from 8 wells as one sample, and the induction of BORIS-specific CTL was confirmed.

The graph of the analysis result using the flow cytometer is represented by a dotted expanded graph, which is a log scale showing the CD8 on the X axis and the HLA- on the Y axis. Fluorescence intensity of tetramer reagent. The numbers in the dotted expansion map indicate (UR+LR) points when the area formed by dividing the expansion map into four is represented as UL (upper left), UR (upper right), LL (lower left), and LR (lower right) The percentage of UR, that is, the percentage of CD8-positive cells that are HLA-tetramer reagent positive.

Figure 20 shows the results of the first stage of the analysis of the sample, which was obtained by culturing the PBMC of the specimen number A2-34 for 13 days using the sequence number 4 of the candidate peptide of BORIS sf6 specific CTL epitope . After confirming the induction of sequence number 4 specific CTL with KLL-Tet, it was detected that the CD8-positive KLL-Tet-positive cell group that was significant in the sample number A2-34 was UR in the 5th (lane) and 11th row in. This incident shows that the peptide of sequence number 4 is a BORIS sf6 specific CTL epitope peptide, and that there is a BORIS specific CTL in the organism of specimen number A2-34.

Figure 21 shows the results of the second stage of line 5 and line 11 where CTL induction was confirmed in the above-mentioned first stage analysis. KLL peptide (SEQ ID NO. 4) specific CTL was detected in well C in row 5 and well F in row 11. This incident proved that the KLL peptide is a BORIS sf6-specific CTL epitope peptide showing HLA-A*02:01 restriction. Since KLL peptide-specific CTL was detected in 2 of 96 wells, the ratio of KLL peptide-specific CTL in peripheral blood PBMC was calculated by the following formula.

Frequency of KLL peptide-specific CTL = (HLA-tetramer reagent positive wells)/(Number of PBMCs used in the experiment×CD8 positive rate before induction)=2/(3×10 ⁷ ×0.16)=4.17× 10 ^-7

Figure 22 shows the analysis results of the first stage of the sample, which was collected from the use of BORIS sf6 specific CTL epitopes Sequence number 5 of the candidate peptide is obtained by culturing the PBMC of specimen number A2-29 for 13 days. After confirming the induction of the specific CTL of sequence number 5 with LLF-Tet, the CD8-positive LLF-Tet-positive cell group that is significant in the sample number A2-29 is detected in the first row, the second row, and the fourth row. , Line 5, Line 6, Line 7, Line 8, Line 9, Line 10, and Line 11 of the UR. This incident shows that the peptide of sequence number 5 is a BORIS sf6-specific CTL epitope peptide, and there is a BOIRS sf6-specific CTL in the organism of specimen number A2-29.

Figures 23-1 and -2 show that the first line, the second line, the fourth line, the fifth line, the sixth line, the seventh line and the eighth line of CTL induction were confirmed in the first stage above. The results of the second stage in rows 9, 10, and 11. Hole B in row 1, hole B and D and G and H in row 2, hole C and F and G in row 4, hole B and F and G in row 5, hole E and hole in row 6 F. Hole F in the 7th row, holes C and G in the 8th row, hole C in the 9th row, hole A in the 10th row, and hole E in the 11th row, the specific LLF peptide (SEQ ID NO: 5) was detected Sexual CTL. This proves that the LLF peptide is a BORIS sf6-specific CTL epitope peptide showing HLA-A*02:01 restriction. Since the LLF peptide-specific CTL was detected in 19 of the 96 wells, the ratio of the LLF peptide-specific CTL in the peripheral blood PBMC was calculated by the following formula.

Frequency of LLF peptide-specific CTL = (number of positive wells of HLA-tetramer reagent)/(number of PBMC used in the experiment × positive rate of CD8 before induction) = 19/(3×10 ⁷ ×0.17)=3.73× 10 ^-6

(6) BORIS sf6-specific CTL induction by PHA blast stimulation

50 mL of peripheral blood was collected from 3 adults who are known to maintain HLA-A*02:01 health, centrifuged, and the plasma portion of the supernatant was collected. Lymphoprep (Axis-Shield PoC As) was used to separate PBMC from the blood cell portion after plasma removal. The separated PBMC was suspended in 10 mL of AIM-V medium, seeded in a culture dish for cell culture, and cultured in a CO ₂ incubator at 37°C for 4 hours. AIM-V medium is meant Life Technologies company in AIM-V, added to a final concentration of 10mM HEPES (Life Technologies, Inc.), 50 μ M final concentration in the medium of 2-mercaptoethanol (Life Technologies, Inc.). After that, only the suspended cells were recovered, using MACS magnetic beads (Miltenyi Biotec), and using magnetic cell separation to separate CD8 positive cells and negative cells.

The CD8-negative cells were suspended in AIM-V medium, and approximately 4×10 ⁵ cells/well were dispensed into a 48-well plate (Corning) for culture.

On the same day that the above-mentioned culture was started, PHA (phytohemagglutinin, Wako) at a final concentration of 1μg/mL and IL-2 (Life Technologies) at a final concentration of 100U/mL were added to the wells of some CD8-negative cells. ). On the 4th day from the start of the culture, the cells were recovered in a culture tube (BD company), suspended in 10 mL of AIM-V medium, and placed in a 75 cm ² culture flask (Nunc company). In addition, IL-2 (Shiono Yoshitaka) was added at a final concentration of 100 U/mL. On the 8th day from the start of the culture, the cells were collected in a culture tube (BD), suspended in 1 mL of AIM-V (Life Technologies), and added with the peptide of SEQ ID NO: 5 (final concentration 20 μg/mL). After that, it was left at room temperature for 1 hour and subjected to 100 Gy radiation treatment to prepare PHA mother cells.

CD8-positive cells are cultured by suspending in AIM-V medium supplemented with human AB serum (LONZA Japan) at a final concentration of 10%, and dispensing approximately 2×10 ⁶ cells/well to a 48-well plate ( Corning).

On the 8th day from the start of culture, 10 ng of IL-7 (R&D Systems) was added to each well. In addition, CD8 positive cells and PHA mother cells were mixed in a 5 to 1 manner, and co-culture was started. From the 9th and 16th day of culture, the preparation of PHA blasts was started again. On the 16th and 23rd of the preparation of PHA blasts, CD8 positive cells and PHA blasts were mixed again in a 5 to 1 manner. , A total of 3 stimulations performed by PHA mother cells. On the 16th day of the culture, 10 U/mL IL-2 (Shiono Yoshitaka) was added to gradually increase the concentration, and finally the concentration was increased to 50 U/mL, and the culture was continued on the 28th day.

The cell group cultured within these 28 days, using the same method as above (5), using PE-labeled HLA-tetramer reagent and The CD8-FITC antibody was stained to confirm the induction of BORIS sf6 specific CTL in the sample.

Figure 24 shows the analysis results of a sample taken from a cell group. The cell group was cultured for 28 days using the sequence number 5 of the candidate peptide of BORIS sf6 specific CTL epitope. to make. After confirming the induction of the sequence number 5 specific CTL with LLF-Tet, a significant CD8-positive LLF-Tet-positive cell group was detected in the sample number A2-S1. This means that the peptide of the LLF peptide (SEQ ID NO: 5) is a BORIS sf6-specific CTL epitope peptide showing HLA-A*02:01 restriction, and it is present in the organism of the specimen number A2-S1 BOIRS specific CTL.

(7) Function analysis of LLF peptide-specific CTL

The function analysis of BOIRS-specific CTL was performed using ELISPOT Set (BD Company). First, collect a part of the cell group induced by BORIS sf6 specific CTL and prepare it to 5×10 ⁵ cells/mL. Disperse this sample at 100 μL/well on the ELISPOST analysis disc in which the anti-IFNγ antibody is immobilized, and place it in a CO ₂ incubator at 37° C. and let it stand for 30 minutes. In this dish, the cells impacted by the peptide of sequence number 5 among the T2 cells were added so that 5×10 ⁴ cells/well were added, and the cells were allowed to stand overnight in a CO ₂ incubator at 37°C. After washing, add biotin-labeled anti-IFNγ antibody and react at room temperature for 2 hours. Wash the reaction solution and add HRP-labeled streptavidin to react. After washing, add 100μL/well of coloring agent, react for 15-30 minutes, and perform photodotization of IFNγ secreted by CTL for measurement.

The results are shown in Figure 25. Compared with the HIV-derived peptide (SLY peptide) with negative control added, or when the peptide was not added (PBS), when stimulated with LLF peptide (SEQ ID NO: 5), more IFNγ was detected obviously Light spot. Therefore, it was confirmed that the LLF peptide (SEQ ID NO: 5) specific CTL that produced IFNγ was induced in the PBMC cultured with the LLF peptide added.

(8) Selection and cultivation of CTL clones

The BORIS sf6 specific CTL and HLA-tetramer reagent, whose IFN γ production ability was confirmed in (7) above, were double-stained with anti-CD8-FITC (MBL company) antibody, and then combined with HLA-tetramer reagent and The cells reacted with the anti-CD8-FITC antibody were seeded into a 96-well plate (Corning) using a flow cytometer. The medium for culturing the cells is made of AIM-V medium with human AB serum (LONZA Japan) added to a final concentration of 10%, penicillin/streptomycin (Life Technologies) with a final concentration of 1%, and a final concentration of 1% the GlutaMAX (Life Technologies, Inc.), a final concentration of 100U / mL of IL-2 (Shionogi Co.), a final concentration of 5 μ g / mL of PHA (Wako Co.) medium. In each well, 50,000 PBMCs obtained by collecting blood from 3 healthy blood donors were added. The PBMCs are cells treated with 100Gy radiation.

In addition, the cells separated by a flow cytometer are used to observe the color of the culture medium and exchange half of the culture medium at any time. And, when the cells are increasing, transfer to a 48-well plate.

The cultured CTL was stained with LLF-Tet and CD8-FITC antibody using the same method as in (5) above, and the result of confirming the increase of CTL is shown in Fig. 26. As a result, a significant CD8-positive LLF-Tet-positive cell group was detected. This means that the monoculture and amplification of CTL specific to the LLF peptide derived from BORIS has been successful.

The cultured CTL was processed in the same procedure as in (7) above, and the result of the functional analysis of the CTL is shown in Fig. 27. As a result, compared with the case where no peptide was added (-), when stimulated with the LLF peptide, more IFNγ spots were detected. Therefore, it was confirmed that the cultured CTL is a CTL specific for the LLF peptide of IFNγ (SEQ ID NO: 5) that is re-stimulated by the LLF peptide.

(9) Function analysis of LLF peptide-specific CTL

The LDH killing assay (TaKaRa Bio) was used to analyze whether the LLF peptide-specific CTLs at the end of (8) culture would attack the cells presenting the LLF peptide. First, prepare the target cell (Target) that becomes the target of the LLF peptide-specific CTL. The following 3 types of cells were prepared: T2 cells that were impacted by the LLF peptide of sequence number 5; as a negative control, cells that were impacted by HIV-derived peptides (SLY peptides) in T2 cells; only T2 cell. The target cells are scattered in a 96-well V-chassis (Corning) per 1×10 ⁴ cells/well. The LLF-specific CTL (Effector) is a cell suspension prepared into a concentration of 9×10 ⁵ cells/mL, 3×10 ⁵ cells/mL, and 1×10 ⁵ cells/mL. Spread 100 μL per well, and spread Mix the target cells in a 96-well plate. After that, the 96-well plate was centrifuged at 1800 rpm for 10 minutes, and then it was allowed to stand in a CO ₂ incubator at 37°C for 4 to 12 hours. Centrifuge the 96-well plate to pellet the cells, and transfer 100 μL of the supernatant to a flat-bottomed 96-well plate. In each well, 100 μL of the reaction solution containing diaphorase was added and allowed to stand at room temperature for 30 minutes, and then the absorbance at 490 nm was measured. With this operation, the LDH generally present in the cell membrane is released outside the cell due to the damage of the cell membrane when the cell is injured. Therefore, the cytotoxicity can be assessed by measuring the amount of LDH in the culture medium. Use this method to explore whether LLF peptide-specific CTL can recognize and attack cells that present the target of LLF peptide.

The results are shown in Figure 28. On the X axis, the ratio of Effector and Target cells is represented by the E/T rate, and on the Y axis is the cytotoxic activity (%). The cytotoxic activity can be calculated according to the formula shown below. It is expressed as: [Sample measured value (Absorbance at 490nm: A490)-Low control (A490)] / [High control (A490)-Low control (A490)] x 100. The high control (A490) means the measurement value when 2% Triton X-100 is added to the target cell suspension, and the low control (A490) means the measurement value when only the target cell suspension is added. The cytotoxic activity (%) value is expressed as the average value of 3 samples.

LLF peptide-specific CTL is compared with when T2 cells pulsed with a negatively controlled HIV-derived peptide (SLY peptide) as target cells, or with T2 cells without peptide added as target cells, When T2 cells pulsed with LLF peptide are used as target cells, they show a high proportion of cytotoxic activity. In other words, it becomes clear that cancer cells exhibiting LLF peptide (SEQ ID NO: 5) will be specifically identified and exert cytotoxic activity.

<Example 4> Discussion on BORIS sf5 (1) Extraction of candidate BORIS-specific HLA-binding epitopes

In the same manner as in Example 3 (3) above, BORIS specific candidate HLA-binding epitopes were extracted. As the target of the analysis, BORIS uses the BORIS B1 isoform (subtype 5), which has the longest amino acid sequence and is also a candidate for BORIS that is specifically expressed in cancer stem cells. About 60% of the retained HLA-A*24:02 candidates with binding epitopes are synthesized. The synthesized peptides are shown below.

[Table 4]

Table 5 shows the characteristics of the synthesized BORIS HLA-A*24:02 binding peptide. It is represented by the short code of the peptide name with 3 or 4 amino acid sequences from the N-terminus of the synthetic peptide. From the left, it shows the peptide name, amino acid sequence, position on the amino acid sequence of BORIS isoform B1, the number of amino acids, and BIMAS (BioInformatics & Molecular Analysis Section, http:/ /thr.cit.nih.gov/index.shtml) HLA Peptide Binding Predictions (http://thr.cit.nih.gov/molbio/hla_bind/). This score is a value that predicts the affinity between HLA-A*24:02 and the peptide. The higher the score, the more likely the HLA and the peptide are to form a stable complex.

[table 5]

(2) Folding test of BORIS specific CTL epitope candidate peptide

The initial stage of the manufacture of the HLA-tetramer reagent starts with the folding of the raw materials, namely HLA, β2 -microglobulin and peptide in an appropriate solution in a test tube. In the folding solution, a triad complex (HLA-monomer) is formed by the association reaction of the three types of raw materials. At this time, if the binding force of HLA and peptide is higher, the association reaction will proceed smoothly. By using a colloidal filter column for analysis, a complex (HLA-monomer) of three types of raw materials can be detected. On the other hand, when HLA has no binding power to peptides, HLA-monomers are hardly detected. Therefore, by analyzing the folding solution over time, or by performing heat treatment, etc., the binding and stability of HLA and peptide can be verified. In this specification, this test is called "folding test".

The 11 types of peptides synthesized above were used to carry out folding tests. In brief, the purified HLA-A*24:02 (60mg/L), β 2-microglobulin (20mg/L) and BORIS specific CTL epitope candidate peptides will be expressed and refined using the E. coli expression system (30μM) added to the folding solution (1M Tris-HCl, 0.5M EDTA, 2M arginine, 5mM GSH, 0.5mM GSSG and protease inhibitor) (the concentrations in parentheses are the final concentration ) And after mixing, divide the folding solution over time and analyze it with a colloidal filter column. A positive control peptide composed of 9 amino acids (SEQ ID NO: 40) and a positive control peptide composed of 10 amino acids (SEQ ID No. 41), and their respective negative control peptides (SEQ ID NO: 42 and 43) As the object of comparison.

The results are shown in Figure 11. According to the results of colloidal filtration column analysis, HLA molecules and β 2-microglobulins can be solubilized with 8M urea when the E. coli expression system is used for expression purification, but the insoluble HLA molecules are not as HLA-single Body formers were detected as agglomerates in 7-8 minutes, followed by the peak of HLA-monomers at around 10 minutes, and β 2-microglobulin at around 14 minutes. The composition or peptide of the folding solution was detected after 15 minutes. Therefore, the result is a bar graph showing the value, which is a value obtained by converting the peak area showing the peak before and after 10 minutes of the formation of HLA-monomer into the estimated HLA-monomer formation amount (mg).

(3) Manufacturing of BORIS specific HLA-tetramer reagent

According to the results of the folding test in (2) above, 10 BORIS-specific CTL epitope candidate peptides other than SEQ ID NO: 11 and HLA-A*24:02 were used to produce PE-labeled HLA-tetramer reagents. In this specification, although the manufactured HLA-tetramer reagents are represented by short codes such as KYA-Tet, this means that HLA-A*24:02 and peptide KYA (KYASVEASKL) and β 2-micro It is a complex of 3 globulins. For simplicity, in the same manner, the above (2) HLA-A * 24 : was added to the refolding solution and mixed 02 β 2- microglobulin and BORIS-specific CTL epitope candidate peptides, a single so HLA- The body is formed. Here, the expression protein was designed by adding a biotin binding site to the C-terminal side of the recombinant HLA-A*24:02 molecule. After the HLA-monomer was formed, biotin was added to this site. The pigment-labeled streptavidin and the above-mentioned biotinylated HLA-monomer are mixed at a molar ratio of 1:4 to obtain an HLA-tetramer reagent.

(4) Induction of BORIS specific CTL

Peripheral blood was collected from 7 adults who were known to maintain HLA-A*24:02 health, and centrifuged at 3,000 rpm for 5 to 10 minutes, and the plasma portion of the supernatant was collected. From the plasma fraction, PBMC was separated by density gradient centrifugation. To each well of a 96-well U-bottom cell culture micro test plate (BECTON DIKINSON), add 10 mL of medium and approximately 3×10 ⁵ PBMCs/well isolated in the above-mentioned PBMC, and suspend them for culture. It is the Hepes modified RPMI1640 medium (Sigma) with 2-mercaptoethanol (final concentration 55μM), L-glutamic acid (final concentration 2mM), streptomycin (final concentration 100μg/mL) and penicillin G as antibiotics (Final concentration 100U/mL) and 5% plasma components. Among them, the above-mentioned BORIS-specific CTL epitope candidate peptides other than SEQ ID NO: 11 were added at a concentration of 10 μg/mL. After 2 days of culture, IL-2 was added at a final concentration of 50 U/mL, and cultured for another 2 weeks.

Culturing the cells for a suitable amount, is added 10 μ L of HLA- PE-labeled tetramer reagent and 20 μ L of CD8-FITC antibody, smoothly mixed, allowed to stand at 4 ℃ 30 minutes. After addition of 1.5 mL of PBS and agitated, centrifuged at 3,000rpm for 5 minutes and the supernatant discarded, the cells were resuspended in 400 μ L of PBS and, using a flow analyzer resolved within 24 hours.

The analysis is carried out in two stages. First, the first stage is to collect the cells of the 8-well column in the 96-well U-bottom cell culture micro-test plate as one sample, and confirm whether the BORIS-specific CTL is induced in this sample. For samples in which CTL induction is confirmed at this stage, perform the second Phase analysis. In the second stage, cells were collected individually from 8 wells as one sample, and the induction of BORIS-specific CTL was confirmed.

Figure 12-1 shows the analysis results of the first stage of the sample, which was collected from the PBMC of the specimen number A24-38 cultured for 13 days using the sequence number 10 of the candidate peptide of BORIS specific CTL epitope By. The figure is represented by a dotted expanded view. The dotted expanded view shows the fluorescence intensity of CD8 on the X axis and the HLA-tetramer reagent on the Y axis on a logarithmic scale. Numbers represent the percentage of UR in (UR+LR) when the area formed by dividing the expanded image into four is represented as UL (upper left), UR (upper right), LL (lower left), and LR (lower right). That is, the proportion of HLA-tetramer-positive cells among CD8-positive cells. After confirming the induction of the specific CTL with sequence number 10 by RMM-Tet, the CD8-positive RMM-Tet-positive cell group was detected in the sample number A24-38 in the second row, fourth row, and eighth row. , UR in line 9. This incident indicates that the peptide with sequence number 10 is a BORIS-specific CTL epitope peptide, and there are BORIS-specific CTLs in organisms with sample numbers A24-38.

Figure 12-2 shows the results of the second stage. The results of the second stage are the further analysis results of the second, fourth, eighth, and 9th lines of the CTL induction confirmed in the first stage . CTL specific to the RMM peptide (SEQ ID NO: 10) was detected in H in row 2, G and H in row 4, F in row 8, and hole A in row 9. This incident proved that the RMM peptide is a BORIS-specific CTL epitope peptide showing HLA-A*24:02 restriction. Since 5 out of 96 wells are detected RMM peptide-specific CTL, so the ratio of RMM peptide-specific CTL in peripheral blood PBMC is calculated by the following formula.

Frequency of RMM peptide-specific CTL = (HLA-tetramer reagent positive wells)/(Number of PBMCs used in the experiment × CD8 positive rate before induction) = 5/(3×10 ⁷ ×0.18)=9.26× 10 ^-7

(5) Preparation of antigen presenting cells

Following the method described in Kuzushima et al., Clin Exp Immunol. 1996; 103: 192-198, an EBV (human herpes virus type IV; Epstein-Barr virus) infection B cell line (hereinafter referred to as EBV infection LCL ( Lymphoblastoid cell line)). To put it simply, the culture supernatant (containing live EBV virus) and PBMC of the EBV production cell line, namely B95-8 cells (obtained from JCRB Cell Bank) and PBMC are mixed to obtain EBV-infected LCL.

(6) Function analysis of RMM peptide-specific CTL

Transfer 1/2 of the PBMC induced in (4) to a 96-well U-bottom cell culture micro-test plate, and add RMM peptide so that the final concentration becomes 100 ng/mL. Then add Brefeldin A (BFA) so that the final concentration becomes 1 μg/mL, and incubate in a 5% CO ₂ thermostat at 37°C for 5 to 16 hours. After culturing, wash the cells, add PE (phycoerythrin) labeled HLA-tetramer reagent and PC5 (phycoerythrin-Cy5) labeled CD8 antibody (Beckman Coulter), and place them at room temperature for 15-30 minutes. After washing, use 4% formaldehyde to fix at 4°C for 15 minutes, then wash with excess detergent. After membrane permeation treatment with 0.1% saponin, FITC-labeled anti-IFNγ antibody (manufactured by MBL) is added and reacted at room temperature for 15-30 minutes. After washing, use a flow cytometer to quantify the rate of IFNγ-positive cells in T cells or the rate of IFNγ-positive cells in HLA-tetramer reagent-positive cells.

The results are shown in Figure 13. Only when stimulated with RMM peptide, IFNγ-positive HLA-tetramer reagent-positive cells appeared in UR, but almost did not appear when RMM peptide was not added. It can be seen that when stimulated with RMM peptides, CTLs that specifically respond to RMM-Tet are induced. Based on this result, the following becomes clear. In PBMC cultured with RMM peptide, the RMM peptide-specific CTL with cytotoxic activity that produces IFNγ is induced by restimulation. This cell Because it is stained by the HLA-tetramer reagent, it is a CTL specific to the peptide derived from HLA-A*24:02 restricted BORIS, that is, the RMM peptide (SEQ ID NO: 10).

(7) BORIS sf5 specific CTL induction

Except that the peptide of sequence number 10 (RMM peptide) was used when preparing PHA mother cells, the same sequence as in Example 3 (6) was used, and it is known that HLA-A*24:02 or HLA-A* At 02:01, healthy adults underwent BORIS sf5-specific CTL induction.

The induced CTL was stained by the same method as in Example 3(5) to confirm whether the BORIS sf5 specific CTL in the sample was induced.

Figure 29 shows the PBMC and RMM peptides (SEQ ID NO: 10) of the specimen number A24-S4 with HLA-A*24:02 or the specimen number A2-S5 with HLA-A*02:01 The results of the analysis of the cell group in which the cells were co-cultured for 28 days. The confirmation of the presence or absence of induction of RMM peptide-specific CTLs in specimen numbers A24-S4 and A2-S5 is to use RMM peptides and use HLA tetramers produced according to the type of HLA possessed by the respective specimens Analytical reagents. As a result, the specimen numbers A24-S4 and A2-S5 both detected significant CD8-positive RMM-Tet-positive cell groups. This incident indicates that the peptide with sequence number 10 is a BORIS sf5-specific CTL epitope peptide, and there are BOIRS-specific CTLs in organisms with sample numbers A24-S4 and A2-S5. It is also known that the peptide of SEQ ID NO: 10 has binding properties to both HLA-type HLA-A*24:02 and HLA-A*02:01.

The functional analysis of BOIRS-specific CTL was performed using the ELISPOT Set (BD) in the same procedure as in Example 3 (7), and the IFNγ secreted by the RMM peptide-specific CTL was measured by photodotization.

Figure 30 shows the result of showing the number of light points by a histogram. And the HIV-derived peptide with negative control added (RYL peptide in the specimen A24-S4, SLY peptide in the specimen A2-S5) When compared with when no peptide is added (PBS), when the RMM peptide is used for stimulation, more light spots are obviously detected.

(8) Analysis of RMM peptides

RMM peptide is the 670~678th amino acid of BORIS B1 isoforms. Here, because BORIS B1 is known to be a homoisomer of 700 amino acids that belongs to BORIS subtype 5, BORIS sf5 has 132 amino acids on the C-terminal side, especially 38 on the C-terminal side. The amino acid (sequence number 2) has a subtype-specific sequence, so the RMM peptide is a sequence specific to BORIS sf5, and is an epitope peptide that can induce CTL. The CTL can also be discovered according to the present invention. Cancer stem cells derived from ovarian cancer that specifically express BORIS sf5 are used as targets.

<Example 5> Discussion on BORIS C1 isoforms (1) Extraction of candidate BORIS-specific HLA-binding epitopes

As the analysis target BORIS, BORIS C1 isoform (subtype 1, SEQ ID NO: 76) was used. Except that HLA-A*02:01 was used as the HLA type, the same procedure was performed as in Example 3 (3). BORIS-specific HLA-binding epitope candidate. The synthesized peptides are shown below.

[Table 6]

Table 7 shows the characteristics of the synthesized BORIS HLA-A*02:01 binding peptide. It is represented by the short code of the peptide name with 3 to 4 amino acid sequences from the N-terminal side of the synthetic peptide. From the left, it is the SYFPEITHI (http://www.syfpeithi.de) that represents the peptide name, amino acid sequence, position on the amino acid sequence of the BORIS C1 isoform, and the number of amino acids for analysis. /0-Home.htm) EPITOPE PREDICTION (http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm) calculated score. This score is used to predict the affinity of HLA and peptide with the structure motif of HLA-A*02:01 molecule and peptide. The higher the score, the more likely HLA and peptide are to form a stable complex. body.

(2) Folding test of BORIS specific CTL epitope candidate peptide

The 11 types of peptides synthesized according to Table 7 were used to perform the folding test. In the folding test, in addition to using HLA-A*02:01 as HLA, the peptides described in Table 7 above were used as epitope candidate peptides, the peptide of SEQ ID NO: 58 was used for the positive control peptide, and the peptide of sequence number 58 was used for the negative control. Except that the peptide of SEQ ID NO: 59 was used as a comparison target, the same method as in Example 4 (2) was used.

The results are shown in Figure 33. The result is the same as that of Example 4 (2), and it is a value converted into an estimated HLA-monomer formation amount (mg). As a result, with the exception of SEQ ID NO: 55, 10 BORIS-specific CTL epitope candidate peptides from SEQ ID NO: 47 to SEQ ID NO. 57 showed sufficient HLA-monomer formation compared with the negative control. That is, it means that in addition to the MAA peptides, the BORIS-specific CTL epitope candidate peptides listed in Table 7 will bind to HLA-A*02:01.

(3) Manufacturing of BORIS specific HLA-tetramer reagent

According to the results of the folding test in (2), except for sequence number 55, except for the use of 10 types of BORIS-specific CTL epitope candidate peptides and HLA-A*02:01 from sequence number 47 to sequence number 57, and In Example 4(3), a PE-labeled HLA-tetramer reagent was produced in the same procedure.

(4) Induction of BORIS specific CTL

Four adults who are known to maintain the health of HLA-A*02:01 were used as subjects, except for sequence number 55, except for the use of BORIS-specific CTL epitope candidate peptides from sequence number 47 to sequence number 57. CTL induction was performed by the same method as in Example 3(5).

For the cell group induced by CTL, the presence or absence of CTL induction was confirmed by the same method as in Example 3 (5). The staining of CTL is carried out using HLA-tetramer reagent. The HLA-tetramer test The agent is a candidate peptide corresponding to the BORIS-specific CTL epitope used in induction. The following shows the representative results of CTL induction.

Figure 34 shows the sample number A2-29, Figure 36 shows the sample number A2-27, and Figure 38 shows the analysis results of the first stage of the sample number A2-34, the sample will be collected PBMC and BORIS-specific CTL epitope candidate peptide sequence number 47 from the above specimen was cultured for 13 days. After confirming the induction of the specific CTL with sequence number 47 using VLE-Tet, a significant CD8-positive cell group with VLE-Tet positive is detected in the sample number A2-29 in the 10th line, and the sample number A2-27 is In line 3, the specimen number A2-34 is in the UR in line 4. This incident shows that the peptide of SEQ ID NO: 47 is a BORIS-specific CTL epitope peptide, and BOIRS-specific is present in the organisms with sample number A2-29, sample number A2-27, and sample number A2-34. Sexual CTL.

Figures 35, 37, and 39 show the results of the second stage where CTL induction was confirmed in the first stage. In Figure 35, it is well H in row 10, in Figure 37, it is well E in row 3, and in Figure 39, the VLE peptide (SEQ ID NO: 47) is detected in well C in row 4 ) Specific CTL. This incident proved that the VLE peptide is a BORIS-specific CTL epitope peptide showing HLA-A*02:01 restriction. Regardless of the specimen, since VLE peptide-specific CTL was detected in 1 of 96 wells, the ratio of VLE peptide-specific CTL in peripheral blood PBMC was calculated by the following formula.

Frequency of VLE peptide-specific CTL = (number of HLA-tetramer reagent positive wells)/(number of PBMCs used in the experiment×CD8 positive rate before induction)=1/(3×10 ⁷ ×0.18)=1.85× 10 ^-7

Figure 40 shows the results of the first stage analysis of the sample, which was collected from the PBMC and BORIS-specific CTL epitope candidate peptide sequence number 48 cultivated for 13 days, and the PBMC was collected for self-examination Body number A2-29. After using KLA-Tet to confirm the induction of the specific CTL of sequence number 48, the significant CD8-positive KLA-Tet-positive cell group was detected in the second, fifth, and 11th lines of the specimen number A2-29 In the UR. This incident indicates that the peptide of SEQ ID NO: 48 is a BORIS-specific CTL epitope peptide, and there are BOIRS-specific CTLs in the organism of specimen number A2-29.

Figure 41 shows the results of the second stage where CTL induction was confirmed in the first stage. In Figure 41, KLA peptide (SEQ ID NO: 48) specific CTLs were detected in well H in row 2, well D in row 5, and well F in row 11. This proves that the KLA peptide is a BORIS specific CTL epitope peptide showing HLA-A*02:01 restriction. Since KLA peptide-specific CTLs were detected in 3 of 96 wells, the proportion of KLA peptide-specific CTLs in peripheral blood PBMC was calculated by the following formula.

Frequency of KLA peptide-specific CTL = (HLA-tetramer reagent positive wells)/(Number of PBMCs used in the experiment × CD8 positive rate before induction) = 3/(3×10 ⁷ ×0.19)=5.26× 10 ^-7

Figure 42 shows the results of the first stage analysis of the sample, which was collected from PBMC and BORIS-specific CTL epitope candidate peptide sequence number 57, which was cultured for 13 days, and the PBMC was collected for self-examination Body number A2-29. After confirming the induction of the specific CTL of SEQ ID NO: 57 using VLT-Tet, a significant CD8-positive VLT-Tet-positive cell group was detected in the UR in the 7th and 9th rows of the specimen No. A2-29. This incident indicates that the peptide of SEQ ID NO: 57 is a BORIS-specific CTL epitope peptide, and that there are BOIRS-specific CTLs in the organism of the specimen number A2-29.

Figure 43 shows the results of the second stage where CTL induction was confirmed in the first stage. In Figure 43, the VLT peptide (SEQ ID NO: 57)-specific CTL was detected in well A in row 7 and well G in row 9. This proves that the VLT peptide is a BORIS-specific CTL epitope peptide showing HLA-A*02:01 restriction. Since VLT peptide-specific CTLs were detected in 2 of 96 wells, the ratio of VLT peptide-specific CTLs in peripheral blood PBMC was calculated by the following formula.

Frequency of VLT peptide-specific CTL = (HLA-tetramer reagent positive wells)/(Number of PBMCs used in the experiment × CD8 positive rate before induction) = 2/(3×10 ⁷ ×0.19)=3.51× 10 ^-7

(5) Function analysis of peptide-specific CTL

PBMC containing KLA peptide-specific CTL induced by specimen A2-29, or PBMC containing VLT peptide-specific CTL induced by specimen A2-29, cultured in 96-well U bottom cells In the micro test plate, each is divided into 2 wells so as to become approximately 3×10 ⁶ pieces. KLA peptide is added to PBMC containing KLA peptide-specific CTL, and VLE peptide is added to PBMC containing VLE peptide-specific CTL, and added to the 2 wells at a final concentration of 100ng/mL 1 hole, the other 1 hole is prepared as an untreated hole. In addition, the anti-CD107a-FITC-labeled antibody and monensin were added to the peptide addition wells and untreated wells, and incubated in a CO ₂ incubator at 37°C for 4 hours. After culturing, wash the cells, add PE-labeled HLA-tetramer reagent corresponding to the respective peptides and PC5 (phycoerythrin-Cy5) labeled anti-CD8 antibody (Beckman Coulter), and place at room temperature for 15-30 minute. Wash with excess detergent, use flow cytometer to detect the degranulation mark of CTL, which is CD107a, to calculate the positive cell rate. It is known that when CTL releases perforin (perforin), granzyme (granzyme) and other cytotoxic factors, it will express CD107a in the inner membrane of intracellular granules on the cell membrane. By detecting CD107a molecules, it can be investigated indirectly Release of cytotoxic factors.

The results are shown in Figure 44 and Figure 45. In Figure 44, CD107a-positive HLA-tetramer reagent-positive cells appeared in UR only when stimulated with KLA peptide, but almost did not appear when KLA peptide was not added. It can be seen that when stimulated with KLA peptides, CTLs that specifically respond to KLA-Tet are induced. Also, in Figure 45, CD107a-positive HLA-tetramer reagent-positive cells appeared in UR only when stimulated with VLT peptides, but almost did not appear when VLT peptides were not added. From this, it can be seen that when stimulated with VLT peptides, CTLs that specifically respond to VLT-Tet are induced. Based on this result, it can be seen that in PBMC cultured with KLA peptide or PBMC cultured with VLT peptide, CTLs with cytotoxic activity that produce granzyme or perforin are induced by restimulation come out. In addition, because this CTL was stained by the HLA-tetramer reagent, it was proved that the peptide derived from HLA-A*02:01 binding BORIS C1 is KLA peptide (SEQ ID NO: 48) or VLT CTL specific for peptide (SEQ ID NO: 57).

The results of the investigation of the induction of HLA-A*02: 01 restricted BORIS-specific CTL are summarized and shown in Table 8.

[Table 8]

(6) Extraction of candidate BORIS specific HLA-A*11:01 binding epitope

In the same manner as in Example 3(3) above, the candidate BORIS-specific HLA-binding epitope was extracted. However, only one point is different. The extracted candidate epitope is HLA-A*11:01, which has the third highest retention rate in Southeast Asia, including the Japanese. The extracted peptide sequences of candidate epitopes were synthesized. The sequences are shown in Table 9-1, and the control peptides are shown in Table 9-2.

Table 9-1 shows the characteristics of the synthesized candidate BORIS specific HLA-A*11:01 binding epitope. For peptides that bind to HLA-A11, it is known that Ile, Met, Ser, Thr, or Val is mostly placed at the second position from the N-terminal, and Lys is mostly placed at the ninth or tenth position. Or any of Arg. In addition, the length of the peptide is well understood to be composed of 9 to 10 amino acids (see Rapin N et al., Curr Protoc Immunol. 2010; Chapter 18: Unit 18.17). It is represented by the short code of the peptide name with 3 to 4 amino acid sequences from the N-terminal side of the synthetic peptide. From the left, it indicates the position of the peptide name, amino acid sequence, BORIS B1 isoform of sf5 and/or C7/C9 isoform of sf6, and the number of amino acids , Use the score calculated by the HLA Peptide Binding Predictions of NetMHC3.4 (http://www.cbs.dtu.dk/services/NetMHC/) for analysis (Nielsen et al., Protein Sci. 2003; 12: 1007 -1017, Lundegaard et al., Nucleic Acids Res. 2008; 36 (Web Server issue): W509-512, Lundegaard et al., Bioinformatics. 2008; 24: 1397-1398 reference). This score is a value that predicts the affinity between HLA-A*11:01 and the peptide. The higher the score, the more likely the HLA and the peptide are to form a stable complex. In addition, the scores of NetMHC 3.4 shown in Tables 9-1 and 9-2, This is a representative example obtained from 11 types of analysis software used for analysis.

(7) Folding test of BORIS specific HLA-A*11:01 restricted CTL epitope candidate peptide

The folding test was performed using 13 types of peptides synthesized according to Table 9-1. For the folding test, in addition to using HLA-A*11:01 as HLA and using the peptides described in Table 9-1 as epitope peptides, the peptide of SEQ ID NO: 73 is used for the positive control peptide and the peptide of sequence number 73 is used for the negative control. Except that the peptide of SEQ ID NO: 40 was used as a comparison target, the same method as in Example 4(2) was used.

The analysis results after 1, 3, and 7 days of the folding test performed on 15 kinds of peptides are shown in Fig. 46. As the positive control peptide, the HLA-A*11:01 restricted peptide derived from CMV pp65 protein (ATV peptide, SEQ ID NO: 73) was used, and as the negative control, HLA-derived from survivin-2B was used. A*24: 02 restricted peptide (AYA peptide, SEQ ID NO: 40) was used as a comparison target. The area representing the peak of HLA-monomer formation is shown as a bar graph. As a result, BORIS-specific CTL epitope candidate peptides (from SEQ ID NO: 60 to SEQ ID NO: 72), compared with the negative control, showed sufficient HLA-monomer formation. That is, it means that the BORIS-specific CTL epitope candidate peptides listed in Table 9-1 will bind to HLA-A*11:01.

(8) Manufacturing of BORIS specific HLA-A*11:01 restricted HLA-tetramer reagent

According to the result of the folding test in (7), a PE-labeled HLA-tetramer reagent was produced in the same procedure as in Example 4 (3). However, 13 types of BORIS-specific CTL epitope candidate peptides ranging from SEQ ID NO. 60 to SEQ ID NO. 72 were used as epitope peptides, and HLA-A*11:01 was used as HLA. As a result, the HLA-tetramer reagent in SEQ ID NO: 68 could not be produced. Therefore, the sequence number 68 was removed, and 12 types of HLA-tetramer reagents with CTL epitope candidate peptides ranging from sequence number 60 to sequence number 72 and HLA-A*11:01 were produced.

(9) Induction of BORIS specific CTL

Two adults who are known to maintain HLA-A*11:01 health were used as subjects, and CTL induction was performed in the same procedure as in Example 3(5). However, as the epitope peptide, 4 types of mixed peptide 1 (SEQ ID NO. 60, SEQ ID NO. 63, and SEQ ID NO: 60) were used, which was a mixture of 4 types of candidate HLA-A*11:01 restricted CTL epitopes. SEQ ID No. 66 and SEQ ID No. 70), or 4 types of mixed peptide 2 (SEQ ID No. 61, SEQ ID No. 64, SEQ ID No. 67 and SEQ ID No. 71), or 4 types of mixed peptide 3 (SEQ ID No. 62, SEQ ID NO. 65, serial number 69 and serial number 72).

The analysis of the cell group induced by the tested CTL is performed in three stages. First, in the first stage, the 96-well U bottom cell culture micro The 8-well cells in the column of the test plate were collected as one sample. Use HLA-tetramer mixing reagent 1 (SVL-Tet, NTH-Tet, KQL-Tet and GLI-Tet) corresponding to the 4 types of mixed peptides used in inducing this sample, or HLA-tetramer Mix reagent 2 (SLA-Tet, CSY-Tet, TVY-Tet and TVL-Tet), or HLA-tetramer mixed reagent 3 (RMS-Tet, GTM-Tet, AAA-Tet and KLLF-Tet), borrow Staining was performed in the same sequence as in Example 3 (5) to confirm whether BORIS-specific CTL was induced. For samples in which CTL induction was confirmed at this stage, the second stage analysis was performed. In the second stage, cells are collected individually from 8 wells as a sample, and this sample is stained with HLA-tetramer mixture reagent 1, 2 or 3 to confirm whether BORIS-specific CTL has been induced. In the third stage, the cells in the wells induced by CTL were confirmed in the second stage. Which of the HLA-tetramer reagents in the HLA-tetramer mixture reagent is reacted with, and the HLA-tetramer reagent is used separately Implement CTL testing. Using this method, it was confirmed that CTL specific to which peptide was induced in the well of the 96-well U-bottomed cell culture micro test plate.

Figure 47 is the analysis result of the first stage. It shows the analysis result of the first stage of the sample. The sample was collected from PBMC with sample number *11-13 and 4 kinds of mixed peptide 1 cultured for 14 days. After staining with HLA-tetramer mixed reagent 1 in the same sequence as in Example 3 (5), a significant CD8-positive HLA-tetramer mixed reagent 1 positive cell group was detected in the first row UR. This result indicates that among the candidate BORIS-specific CTL epitopes Among them, there is at least one type of CTL epitope, and there are BORIS-specific CTLs in the organism of the specimen number A*11-13.

Figure 48 shows the results of the second stage analysis in the first row where CTL induction was confirmed in the first stage of the analysis. In the well B (1-B) of the first row, the induction of CTL specific to the HLA-A*11:01 binding BORIS specific CTL epitope was confirmed.

Figure 49 shows the results of the third-stage analysis of well B in the first row where CTL induction was confirmed in the second-stage analysis. As a result, CTL was confirmed only in the case of staining with KQL-Tet. This proves that the KQL peptide (SEQ ID NO: 66) is an HLA-A*11:01 restricted BORIS specific CTL epitope peptide.

Figure 50 is the analysis result of the first stage. It shows the analysis result of the first stage of the sample, which was collected from PBMC with sample number *11-13 and 4 types of mixed peptide 2 cultured for 14 days. After using HLA-tetramer mixed reagent 2 and stained by the same sequence as in Example 3 (5), a significant CD8-positive HLA-tetramer mixed reagent 2 positive cell group was detected in line 12 UR. This result indicates that among the candidate BORIS-specific CTL epitopes used for the study, there is at least one type of CTL epitope, and there are BORIS-specific epitopes in the organisms of the specimen number A*11-13. CTL.

Figure 51 shows the results of the second-stage analysis on line 12 where CTL induction was confirmed in the first-stage analysis. In well E (12-E) in row 12, the induction of CTL specific to the HLA-A*11:01-binding BORIS-specific CTL epitope was confirmed.

Figure 52 shows the results of the third stage analysis of the hole E in the 12th row where CTL induction was confirmed in the second stage of the analysis. CTL was confirmed only in the case of staining with TVY-Tet. This proves that TVY peptide (SEQ ID NO: 67) is an HLA-A*11:01 restricted BORIS-specific CTL epitope peptide.

Figure 53 is the analysis result of the first stage. It shows the analysis result of the first stage of the sample, which was collected from PBMC with sample number *11-16 and 4 kinds of mixed peptide 3 cultured for 14 days. After staining with the HLA-tetramer mixed reagent 3 in the same sequence as in Example 3 (5), a significant CD8-positive HLA-tetramer mixed reagent 3 positive cell group was detected in the 7th row and UR on line 11. This result indicates that among the candidate BORIS-specific CTL epitopes used in the study, there is at least one type of CTL epitope, and there are BORIS-specific epitopes in the organisms of the specimen number A*11-16. CTL.

Figure 54 shows the results of the second-stage analysis on the 7th and 11th lines where CTL induction was confirmed in the first stage of the analysis. In hole E (7-E) in row 7 and hole H (11-H) in row 11, one of the CTLs specific to the HLA-A*11:01 restricted BORIS-specific CTL epitope was confirmed Induce.

Figure 55 shows the results of the third-stage analysis of the hole E in the seventh row and the hole H in the 11th row where CTL induction was confirmed in the second-stage analysis. Hole E in row 7 is only for staining with GTM-Tet, and hole H in row 11 is for staining only with KLLF-Tet Confirm to the CTL. This proves that GTM peptide (SEQ ID NO: 65) and KLLF peptide (SEQ ID NO: 72) are HLA-A*11:01 restricted BORIS-specific CTL epitope peptides.

<Example 6> Preparation of BORIS sf5 and sf6 specific antibodies

For the purpose of obtaining BORIS sf5 and BORIS sf6 specific antibodies, a peptide sequence composed of 8-20 amino acids was synthesized from sequence number 1 and sequence number 2, respectively, and added to the C-terminus or N-terminus of the peptide To cysteine residues, KHL (Keyhole limpet hemocyanin) is combined and used as an immunogen according to a general method. Rabbits and guinea pigs were immunized 4-6 times every other week or every week. After the immunization, blood is collected from each individual, and the blood is purified using affinity columns made of peptides for immunogens to obtain specific antibodies.

The verification of specificity is carried out using a cell extract of 293T cells. The cell extract of 293T cells includes the cell extract of cultured cells 293T and untreated 293T cells, which make BORIS sf5 and BORIS sf6 transiently express respectively. Extract. A Myc Tag sequence was added to the transient BORIS sf5 and BORIS sf6 genes, and a specific Myc Tag antibody was used as a positive control for this.

As shown in Figure 56, the BORIS sf5 and BORIS sf6 specific antibodies obtained from the verification results of ink spotting in the West, when the respective BORIS expression 293T cell extracts were used, both displayed and displayed in the positive control station. The detected band is detected at the position of the same molecular size In contrast, no band was detected in the untreated 293T cell extract. Therefore, it is considered that antibodies that specifically bind to BORIS sf5 and BORIS sf6 can be obtained. In addition, the tissue sections of the cancer-affected part resected from a lung cancer patient were stained with the BORIS sf5 specific antibody, and it is clear that there are cases where it is judged as negative and some cases are judged as positive (see Figure 57). By using the obtained specific antibodies, the performance of BORIS sf5 and BORIS sf6 can be confirmed, showing that it can be a powerful tool for judging the suitability of peptide vaccine therapy, etc. The peptide vaccine therapy uses the one identified in the present invention. CTL epitope peptide.

<Example 7> Preparation of siRNA against BORIS sf6 (1) siRNA design

The BORIS-targeted siRNA designed to have the sequence shown in the following table were transfected into MS751 and CaSki, respectively, according to the experimental specifications described in the manufacturer's instruction manual using Lipofectamine RNAiMAX. The cells were resolved 48 hours after transfection. As a negative control, Trilencer-27 Universal Scrambled Negative Control siRNA (SR30004, Origene) was used.

[Table 10]

The results of gene knockdown are shown in Figures 14 and 15. Figure 14-1 (a) shows that the performance level of BORIS (B0) is significantly suppressed by three types of siRNA (quantitative RT-PCR). In addition, from Fig. 14-1(b) and Fig. 14-2, some cell growth inhibition was observed in siRNA1 and siRNA2. It is thought that the greater the number of BORIS subtypes that may be inhibited, the higher the proliferation inhibition effect will be. However, in siRNA1 and siRNA2, the suppression of stem cell genes did not occur at all (Figure 15).

In addition, as shown in Figures 16-1 and 16-2, the sphere-forming ability of CasKi and MS751 was significantly inhibited by siRNA2 (the results of cell growth inhibition are not necessarily consistent).

Moreover, gene knockdown of siRNA1~3 has nothing to do with radiation tolerance (Figure 17).

From Figure 18, it can be seen that if the BORIS performance is high, the survival rate will be significantly reduced. Therefore, BORIS has been recognized as a meaningful poor prognostic factor.

[Industrial Utilization]

According to the present invention, it is possible to provide therapeutic agents effective for various cancers. In particular, CTLs induced by the epitope peptides of the present invention can induce CTLs capable of specifically attacking various cancer cells, especially cancer stem cells that are considered to be the cause of malignant tumors, and have fewer side effects and high effects. Anticancer agents are very useful.

<110> Hokkaido Public University Corporation Sapporo Medical University Institute of Medical Biology Co., Ltd.

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<212> PRT

<213> Artificial sequence

<220>

<223> Tumor antigen peptide

<400> 48

<210> 49

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 49

<210> 50

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 50

<210> 51

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 51

<210> 52

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 52

<210> 53

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 53

<210> 54

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 54

<210> 55

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Candidate HLA-A02: 01 binding peptide

<400> 55

<210> 56

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 56

<210> 57

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Tumor antigen peptide

<400> 57

<210> 58

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 58

<210> 59

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 59

<210> 60

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 60

<210> 61

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 61

<210> 62

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 62

<210> 63

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 63

<210> 64

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 64

<210> 65

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Tumor antigen peptide

<400> 65

<210> 66

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Tumor antigen peptide

<400> 66

<210> 67

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Tumor antigen peptide

<400> 67

<210> 68

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> HLA-A11: 01 binding peptide

<400> 68

<210> 69

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 69

<210> 70

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 70

<210> 71

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 71

<210> 72

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Tumor antigen peptide

<400> 72

<210> 73

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CTL epitope candidate peptide

<400> 73

<210> 74

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 74

<210> 75

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 75

<210> 76

<211> 663

<212> PRT

<213> Homo (Homo sapiens)

<400> 76

Claims (15)

A method for inducing cytotoxic T cells. The cytotoxic T cells will specifically identify cells that exhibit the Brother of the Regulator of Imprinted Sites (BORIS) gene, and the BORIS gene belongs to isoform A Or C, or subtype 5 or 6, the method includes contacting any one of the following (a) or (b) with peripheral blood lymphocytes in vitro : (a) Polypeptide, which is The partial peptides of the aforementioned BORIS protein are represented by any one of sequence numbers 3, 4, 5, 10, 47, 65, 66, 67, and 72, and have HLA binding ability; (b) polynucleotides, which At least one of the polypeptides described in (a) above is encoded. A polypeptide, which is represented by any one of sequence numbers 3, 4, 5, 10, 47, 65, 66, 67, and 72, and has HLA binding ability. A polypeptide, which is used in the method described in claim 1, is represented by any one of sequence numbers 3, 4, 5, 10, 47, 65, 66, 67 and 72 and has HLA binding ability. A polypeptide, which is used in the method described in claim 1 and has HLA-A11 antigen binding ability, and is represented by sequence number 65, 66, 67 or 72. A polypeptide for use in the method described in claim 1 Among them, it has HLA-A2 antigen binding ability, which is represented by SEQ ID NO: 4, SEQ ID No. 5, SEQ ID No. 10, or SEQ ID NO: 47. A polypeptide, which is used in the method described in claim 1, and has HLA-A24 antigen binding ability, and is represented by sequence number 3 or 10. A polynucleotide used in the method according to claim 1 to encode the polypeptide according to any one of claims 3 to 6. A cytotoxic T cell inducer comprising at least one of the polypeptides described in any one of claims 3 to 6 as an active ingredient. A pharmaceutical composition comprising the cytotoxic T cell inducer described in claim 8 as an active ingredient. A composition for the treatment of cancer stem cells comprising a polynucleotide having a sequence complementary to the polynucleotide described in claim 7 as an active ingredient. An expression vector, which contains the polynucleotide as described in claim 7. An HLA-tetramer containing the polypeptide and HLA as described in claim 2. A method for producing antigen-presenting cells, which comprises the steps of contacting the following (a) or (b) with cells having antigen-presenting ability in a test tube: (a) The polypeptide according to claim 2; (b) a polynucleotide, which encodes at least one of the polypeptides described in (a) above. An antibody that specifically binds to at least a part of the polypeptide represented by any of SEQ ID NOs: 3, 4, 5, 10, 47, 65, 66, 67, and 72. A kit for detecting BORIS protein, which comprises the antibody according to claim 14.

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